US9534227B2 - Methods for high yield production of terpenes - Google Patents

Methods for high yield production of terpenes Download PDF

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US9534227B2
US9534227B2 US13/893,105 US201313893105A US9534227B2 US 9534227 B2 US9534227 B2 US 9534227B2 US 201313893105 A US201313893105 A US 201313893105A US 9534227 B2 US9534227 B2 US 9534227B2
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plant
synthase
promoter
limonene
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Toni Kutchan
Yasuhiro Higashi
Xiaohong Feng
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Donald Danforth Plant Science Center
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C07K2319/08—Fusion polypeptide containing a localisation/targetting motif containing a chloroplast localisation signal

Definitions

  • the present invention relates to the enhanced production and accumulation of terpenes in plants via the expression of fusion proteins comprising various combinations of geranyl diphosphate synthase large and small subunits with limonene synthase.
  • the present invention also relates to engineering of oilseed plants, exemplified by camelina , to accumulate monoterpene and sesquiterpene hydrocarbons, exemplified herein by the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon 5-epi-aristolochene. This establishes a framework for the rapid engineering of oilseed crop production platforms for terpene-based biofuels.
  • Jet fuel is a mixture of many different hydrocarbons. Modern analytical techniques indicate that there may be a thousand or more. The range of their sizes (carbon numbers) is restricted by specific physical requirements of a specific jet fuel product.
  • Kerosine-type jet fuel has a carbon number distribution between about 8 and 16 carbons. Most of the hydrocarbons in jet fuel are members of the paraffin, naphthene and aromatic classes. The compounds that boil near the middle of the kerosine-type jet fuel boiling-range are C10 aromatics, C11 naphthenes, and C12 waxes.
  • Plants synthesize a wide repertoire of cyclic and linear low molecular weight hydrocarbon compounds, which have the potential to be readily converted into jet fuel and industrial solvents.
  • the cyclic monoterpene, limonene, (4S)-1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene) occurs naturally in various ethereal oils, particularly oils of lemon, orange, caraway, dill and bergamot, and is a valuable industrial chemical.
  • Some limonene is prepared by extraction from plants of the mint family, a large quantity is obtained from citrus oils, which are typically 80-90% limonene, and some is obtained from pine oil.
  • C-10 terpenes are synthesized in plastids of specialized gland cells (Turner et al., (1999). Plant Physiology 120: 879-886) from precursors derived via the non-mevalonate pathway from pyruvate and glyceraldehyde-3-phosphate (Rohdich et al., Current Opinion in Chemical Biology 5: 535-540).
  • C-15 terpenes (sesquiterpenes) are synthesized in the cytosol via the mevalonate pathway from acetyl-CoA (Chappell, J (2004) Trends in Plant Science. 9: 266-269). The volatile products of mono- and sesquiterpene biosynthesis in most plants are either secreted into specialized storage cavities or are released to the atmosphere.
  • the first committed step of monoterpene is mediated Geranyl diphosphate synthase (GDS) which catalyzes the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to form GPP, the immediate acyclic precursor of monoterpenes.
  • GPP is converted to ( ⁇ )-4S-limonene by the catalytic action of ( ⁇ )-4S-limonene synthase (cyclase), which represents the primary precursor of various monoterpenes including its downstream metabolites ( ⁇ )-trans-carveol and carvone; as well as the precursor of S-linalool.
  • FIG. 1 Wise et al. (1997) In “Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2” (Cane, D. E., ed.), Elsevier Science, Oxford (1998).
  • GPP synthase and 4S-limonene synthase has been isolated from several plant sources, including grape, geranium, sage (Croteau et al. (1989) Arch. Biochem. Biophys. 271:524-535; Heide et al. (1989) Arch. Biochem. Biophys. 273:331-338; Suga et al. (1991) Phytochemistry 30:1757-1761; Clastre et al. (1993) Plant Physiol. 102:205-211); and spearmint (Colby et al., (1993) J. Biol. Chem. 268(31) 23016-23024) and various cDNA clones are publicly available.
  • Camelina As Arabidopsis , it can be readily transformed by floral dip. Camelina is not a foodstuff plant and grows on marginal lands (e.g. Montana) that are generally considered unsuitable for large scale food production. Camelina is being investigated as a winter crop for southern Missouri and could potentially be double-cropped with soy. These characteristics make Camelina an ideal candidate plant to be developed as a chemical factory, particularly if high level production and accumulation of chemicals can be demonstrated in seeds. It is believed, however that the successful large scale biosynthesis and production of terpenes in Camelina seed has not been previously reported.
  • the current invention is based, at least in part, on the surprising discovery that the over expression of fusion proteins comprising either the GPP synthase large and small subunits, and limonene synthase, or one or more of these subunits fused to limonene synthase, in Camelina seeds results in the high level production and stable accumulation of various terpenes within the seeds.
  • the present invention also surprisingly demonstrates that plants, in particular oil seed crops, can produce and accumulate monoterpene and sesquiterpene hydrocarbons in seeds.
  • the resulting transgenic plants provide for the first time a viable approach for the large scale commercial production of commercially important terpenes in plants, with the potential to directly provide a renewable source of aromatic hydrocarbons, suitable for use for the production of jet fuel, organic solvents, plastics and high value industrial raw materials.
  • the invention includes a transgenic plant comprising a heterologous nucleic acid sequence comprising a method for the production of a monoterpene, comprising the steps of:
  • the geranyl diphosphate synthase small subunit comprises an amino acid sequence selected from Table D1.
  • the geranyl diphosphate synthase large subunit comprises an amino acid sequence selected from Table D2.
  • the limonene synthase comprises an amino acid sequence selected from Table D3.
  • the method further comprises regenerating stably transformed transgenic plants.
  • the terpene is limonene.
  • the plant cell is co-transformed.
  • the first and second expression control sequences comprise constitutive promoters.
  • first and second expression control sequences comprise cell type specific promoters.
  • the first and second expression control sequences comprise seed specific promoters.
  • the first set of expression control sequences comprises the soybean oleosin promoter, and soybean oleosin terminator.
  • the second set of expression control sequences comprises the rapeseed napin promoter and soybean glycinin terminator.
  • the first and second set of expression control sequences comprises the RuBisCo small subunit transit peptide.
  • the plant cell is derived from a monocotyledonous plant. In some embodiments, the plant cell is derived from a dicotyledonous plant. In some embodiments the plant cell is derived from a plant that naturally produces a terpene. In some embodiments, the plant cell is derived from Camelina sativa . In some embodiments, the method further comprises the step of growing the transgenic plant, and harvesting the seeds. In some embodiments, the plant has a seed terpene content of at least 1.0 mg/g dry weight.
  • the current invention includes a method for the production of a terpene, comprising the step of:
  • the geranyl diphosphate synthase small subunit comprises an amino acid sequence selected from Table D1.
  • the geranyl diphosphate synthase large subunit comprises an amino acid sequence selected from Table D2.
  • the limonene synthase comprises an amino acid sequence selected from Table D3.
  • the method further comprises regenerating stably transformed transgenic plants.
  • the terpene is limonene.
  • the plant cell is co-transformed.
  • the first and second expression control sequences comprise constitutive promoters.
  • first and second expression control sequences comprise cell type specific promoters.
  • the first and second expression control sequences comprise seed specific promoters.
  • the first set of expression control sequences comprises the soybean oleosin promoter, and soybean oleosin terminator.
  • the second set of expression control sequences comprises the rapeseed napin promoter and soybean glycinin terminator.
  • the first and second set of expression control sequences comprises the RuBisCo small subunit transit peptide.
  • the plant cell is derived from a monocotyledonous plant. In some embodiments, the plant cell is derived from a dicotyledonous plant. In some embodiments the plant cell is derived from a plant that naturally produces a terpene. In some embodiments, the plant cell is derived from Camelina sativa . In some embodiments, the method further comprises the step of growing the transgenic plant, and harvesting the seeds. In some embodiments, the plant has a seed terpene content of at least 1.0 mg/g dry weight.
  • Certain embodiments include a fusion protein comprising geranyl diphosphate synthase large subunit fused in frame to geranyl diphosphate synthase small subunit.
  • the geranyl diphosphate synthases are selected from an amino acid sequence as set forth in Tables D1 or D2.
  • the fusion protein is characterized by an improved rate of geranyl diphosphate production in vivo compared to the separate expression of the geranyl diphosphate synthase large and small subunits under comparable expression levels and incubation conditions.
  • Certain embodiments include a fusion protein comprising a geranyl diphosphate synthase large or small subunit is fused in frame to limonene synthase.
  • the fusion protein is characterized by an improved rate of limonene synthesis compared to a mixture of geranyl diphosphate synthase and limonene synthases at the same molar concentration, and incubated under comparable reaction conditions.
  • the geranyl diphosphate synthase is selected from an amino acid sequence as set forth in Tables D1 or D2
  • the limonene synthase is selected from an amino acid sequence as set forth in Table D3.
  • Certain embodiments include a comprising a geranyl diphosphate synthase large subunit fused in frame to a geranyl diphosphate synthase small subunit fused in frame to limonene synthase.
  • the fusion protein is characterized by an improved rate of limonene synthesis compared to a mixture of geranyl diphosphate synthase and limonene synthases at the same molar concentration, and incubated under comparable reaction conditions.
  • the geranyl diphosphate synthase is selected from an amino acid sequence as set forth in Tables D1 or D2
  • the limonene synthase is selected from an amino acid sequence as set forth in Table D3.
  • Certain embodiments include an expression vector comprising a polynucleotide sequence encoding a fusion protein of any of foregoing fusion proteins.
  • Certain embodiments include a transgenic plant comprising within its genome
  • the geranyl diphosphate synthase small subunit comprises an amino acid sequence selected from Table D1.
  • the geranyl diphosphate synthase large subunit comprises an amino acid sequence selected from Table D2.
  • the limonene synthase comprises an amino acid sequence selected from Table D3.
  • the terpene is limonene.
  • the first and second expression control sequences comprise constitutive promoters.
  • the first and second expression control sequences comprise cell type specific promoters.
  • the first and second expression control sequences comprise seed specific promoters.
  • the first set of expression control sequences comprises the soybean oleosin promoter, and soybean oleosin terminator.
  • the second set of expression control sequences comprises the rapeseed napin promoter and soybean glycinin terminator.
  • the first and second set of expression control sequences comprises the RuBisCo small subunit transit peptide.
  • the plant cell is derived from a monocotyledonous plant. In some aspects of the transgenic plant, the plant cell is derived from a dicotyledonous plant. In some aspects of the transgenic plant, the plant cell is derived from a plant that naturally produces a terpene. In some aspects of the transgenic plant, the plant cell is derived from the genus Camelina . In some aspects of the transgenic plant, the transgenic plant has a seed monoterpene content of at least 1.0 mg/g dry weight.
  • Certain embodiments include a transgenic plant comprising within its genome, a first nucleotide sequence encoding a fusion protein comprising a geranyl diphosphate synthase small subunit or a geranyl diphosphate synthase large subunit fused in frame to a limonene synthase, operatively linked to a first set of expression control sequences that drive expression of the geranyl diphosphate fusion protein in the plant cell;
  • fusion protein is expressed primarily in the plant cell plastids.
  • the geranyl diphosphate synthase small subunit comprises an amino acid sequence selected from Table D1.
  • the geranyl diphosphate synthase large subunit comprises an amino acid sequence selected from Table D2.
  • the limonene synthase comprises an amino acid sequence selected from Table D3.
  • the terpene is limonene.
  • the first and second expression control sequences comprise constitutive promoters.
  • the first and second expression control sequences comprise cell type specific promoters.
  • the first and second expression control sequences comprise seed specific promoters.
  • the first set of expression control sequences comprises the soybean oleosin promoter, and soybean oleosin terminator.
  • the second set of expression control sequences comprises the rapeseed napin promoter and soybean glycinin terminator.
  • the first and second set of expression control sequences comprises the RuBisCo small subunit transit peptide.
  • the plant cell is derived from a monocotyledonous plant. In some aspects of the transgenic plant, the plant cell is derived from a dicotyledonous plant. In some aspects of the transgenic plant, the plant cell is derived from a plant that naturally produces a terpene. In some aspects of the transgenic plant, the plant cell is derived from the genus Camelina . In some aspects of the transgenic plant, the transgenic plant has a seed monoterpene content of at least 1.0 mg/g dry weight.
  • the transgenic plant has a seed monoterpene content of at least 1.2 mg/g dry weight. In some aspects of the transgenic plant, the transgenic plant has a seed monoterpene content of at least 1.4 mg/g dry weight. In some aspects of the transgenic plant, the transgenic plant has a seed monoterpene content of at least 1.6 mg/g dry weight. In some aspects of the transgenic plant, the transgenic plant has a seed monoterpene content of at least 1.8 mg/g dry weight. In some aspects of the transgenic plant, the transgenic plant has a seed monoterpene content of at least 2.0 mg/g dry weight.
  • the present invention provides the following:
  • a method for the production of a terpene comprising the steps of:
  • fusion protein is operatively linked to a set of expression control sequences that drive expression of the fusion protein in the plant cell;
  • fusion protein is primarily expressed in a plastid of the plant cell.
  • the method of 30, further comprising the step of growing the transgenic plant, and harvesting the seeds.
  • a fusion protein comprising geranyl diphosphate synthase large subunit fused in frame to geranyl diphosphate synthase small subunit.
  • 35 The fusion protein of 34, wherein the fusion protein is characterized by an improved rate of geranyl diphosphate production in vivo compared to the separate expression of the geranyl diphosphate synthase large and small subunits under comparable expression levels and incubation conditions.
  • 36 A fusion protein comprising a geranyl diphosphate synthase large or small subunit fused in frame to limonene synthase.
  • 37. The fusion protein of 36, wherein the fusion protein is characterized by an improved rate of limonene synthesis compared to a mixture of geranyl diphosphate synthase and limonene synthases at the same molar concentration, and incubated under comparable reaction conditions. 38.
  • the fusion protein of 37 wherein the geranyl diphosphate synthase is selected from an amino acid sequence as set forth in Tables D1 or D2, and the limonene synthase is selected from an amino acid sequence as set forth in Table D3.
  • a fusion protein comprising a geranyl diphosphate synthase large subunit fused in frame to a geranyl diphosphate synthase small subunit fused in frame to limonene synthase. 40.
  • the fusion protein of 39 wherein the fusion protein is characterized by an improved rate of limonene synthesis compared to a mixture of geranyl diphosphate synthase and limonene synthases at the same molar concentration, and incubated under comparable reaction conditions.
  • An expression vector comprising a polynucleotide sequence encoding a fusion protein of any of 33 to 41. 43.
  • the expression vector of 42 wherein the geranyl diphosphate synthase is selected from an amino acid sequence as set forth in Tables D1 or D2, and the limonene synthase is selected from an amino acid sequence as set forth in Table D3. 44.
  • a transgenic plant comprising within its genome,
  • FIG. 1 Shows some representative exemplary terpenes of the invention.
  • FIG. 2 Shows the synthetic scheme through which geranyl diphosphate synthase (GDS) and limonene synthase (LS) catalyze the production of Limonene from IPP and DMAPP.
  • GDS geranyl diphosphate synthase
  • LS limonene synthase
  • FIG. 3A Shows the SDS PAGE analysis of the recombinant production of geranyl diphosphate synthase (GDS) and limonene synthase (LS) in E. coli .
  • NC vector control
  • GSL geranyl diphosphate synthase large subunit
  • GSS geranyl diphosphate synthase small subunit
  • LS limonene synthase.
  • FIG. 3B shows GDS in vitro enzyme reactions analyzed by GS-MS.
  • Substrates IPP and DMAPP were incubated with, E. coli recombinant GDS extract (spectra 1), boiled E. coli recombinant GDS extract (spectra 2), and geranyl diphosphate (GPP) (spectra 3); then the resulting GPP was hydrolyzed by alkaline phosphatase to produce geraniol.
  • GPP geranyl diphosphate
  • FIG. 3C shows, LS in vitro enzyme reactions which were analyzed by GS-MS.
  • Substrate GPP was incubated with, E. coli recombinant LS extract (spectra 1), boiled E. coli recombinant LS extract (spectra 2), and limonene (spectra 3).
  • FIG. 4 Shows the accumulation of limonene detected by GC-MS.
  • A T2 Camelina seed extract expressing the individual enzymes GDS and LS in plastids using the TPGDSTPLS vector (#3-5), B, wild-type Camelina seed extract. Peak 1, C 10 H 16 ; Peak 2, C 10 H 16 ; Peak 3, C 10 H 16 ; Peak 4, internal standard; Peak 5, limonene (C 10 H 16 ); Peak 6, C 10 H 16 O; Peak 7, C 10 H 14 O.
  • FIG. 5 Shows the limonene contents of T3 homozygous seeds expressing the individual enzymes GDS and LS in plastids using TPGDSTPLS vector. Ten seeds from each T3 line were analyzed by GC-MS. Bars show SD values from 3 to 6 extractions.
  • FIG. 6 Shows the results of Genomic DNA PCR analysis for the monoterpene genes from 10-day-old T2 leaves.
  • PCR templates Lane 1, genomic DNA extracted from T2 plants expressing the individual enzymes GDS and LS in plastid using TPGDSTPLS vector; Lane 2, genomic DNA extracted from T2 plants expressing the individual enzymes GDS and LS in cytosol using GDSLS vector; Lane 3, wild-type Camelina genomic DNA.
  • FIG. 7 Shows the results of Expression analysis (RT-PCR) for the monoterpene genes in T2 developing seeds.
  • PCR templates Lane 1, genomic DNA extracted from T2 seeds expressing the individual enzymes GDS and LS in plastid using TPGDSTPLS vector; Lane 2, genomic DNA extracted from T2 seeds expressing the individual enzymes GDS and LS in cytosol using GDSLS vector; Lane 3, wild-type Camelina genomic DNA.
  • FIG. 8 Shows the results of GS-MS analysis of samples from T2 mature seeds transformed with GDS and LS after in vitro coupling enzyme reactions.
  • Substrates IPP and DMAPP were incubated with, seed extract expressing the individual enzymes GDS and LS in plastid using TPGDSTPLS vector (spectra 1), seed extract expressing the individual enzymes GDS and LS in cytosol using GDSLS vector (spectra 2), wild-type seed extract (spectra 3), wild-type seed extract and both E. coli recombinant GDS and LS (spectra 4).
  • FIG. 9 shows the results of SDS-PAGE analysis of E. coli expressed recombinant proteins comprising N-terminal His-tagged versions of 2 fusion proteins comprising from the N-terminus, the GDS small subunit and large subunit fused to LS via a 9 amino acid-linker and a fusion protein comprising the same proteins but in the opposite orientation.
  • FIG. 9B shows E. coli recombinant GDS and LS in vitro coupling enzyme reactions which were analyzed by GS-MS. Substrates IPP and DMAPP were incubated with, E. coli recombinant GDS9aaLS extract (spectra 1), boiled E.
  • FIG. 9C Shows the results of GS-MS analysis of samples of, E. coli -expressed recombinant proteins incubated with substrates IPP and DMAPP. The results with LS9aaGDS extract (spectra 1), boiled E. coli recombinant LS9aaGDS extract (spectra 2), and limonene (spectra 3).
  • FIG. 10 Shows an exemplary E. coli expression vector (GSS pET28) for geranyl diphosphate synthase small subunit (GSS).
  • FIG. 11 Shows an exemplary E. coli expression vector (GSL pET28) for geranyl diphosphate synthase large subunit (GSL).
  • FIG. 12 Shows an exemplary E. coli expression vector (pET28-GDS) for geranyl diphosphate synthase (GDS) expressing a fusion protein of small subunit (GSS) and large subunit (GSL).
  • pET28-GDS geranyl diphosphate synthase
  • GDS geranyl diphosphate synthase
  • GSS small subunit
  • GSL large subunit
  • FIG. 13 Shows an exemplary E. coli expression vector (LSfull pET28) for limonene synthase (LS) full-length cDNA.
  • FIG. 14 Shows an exemplary E. coli expression vector (pET28-LS) for limonene synthase (LS).
  • FIG. 15 Shows an exemplary E. coli cloning vector (pNapin) with an AscI site for preparing binary vectors as described herein.
  • FIG. 16 Shows an exemplary E. coli cloning vector (pNaMluI) with a MluI site for preparing binary vectors as described herein.
  • FIG. 17 Shows an exemplary E. coli cloning vector (pNaMluIOleosin) with an oleosin promoter and an oleosin terminator.
  • FIG. 18 Shows an exemplary E. coli cloning vector (pNaAscINapin) with a napin promoter and a glycinin terminator.
  • FIG. 19 Shows an exemplary E. coli cloning vector (putative peaRubiscoS CDS+intro pET28) with a putative RuBisCO small subunit.
  • FIG. 20 Shows an exemplary E. coli cloning vector (pNaMluIOleosinTP) with an oleosin promoter, a RuBisCO transit peptide and an oleosin terminator.
  • pNaMluIOleosinTP E. coli cloning vector with an oleosin promoter, a RuBisCO transit peptide and an oleosin terminator.
  • FIG. 21 Shows an exemplary E. coli cloning vector (pNaAscINapinTP) with a napin promoter, a RuBisCO transit peptide and a glycinin terminator.
  • FIG. 22 Shows an exemplary E. coli cloning vector (pNaMluIOleosinTPGDS) with an oleosin promoter, a RuBisCO transit peptide, GDS and an oleosin terminator.
  • pNaMluIOleosinTPGDS E. coli cloning vector with an oleosin promoter, a RuBisCO transit peptide, GDS and an oleosin terminator.
  • FIG. 23 Shows an exemplary E. coli cloning vector (pNaAscINapinTPLS) with a napin promoter, a RuBisCO transit peptide, LS and a glycinin terminator.
  • FIG. 24 Shows an exemplary plant expression vector (pRSe2) with a cytomegalovirus (CMV) promoter, a Discosoma red fluorescent protein (DsRed) and a nopaline synthase (NOS) terminator.
  • pRSe2 cytomegalovirus
  • CMV cytomegalovirus
  • DsRed Discosoma red fluorescent protein
  • NOS nopaline synthase
  • FIG. 25 Shows an exemplary plant expression vector (TPGDSTPLS) for co-expressing geranyl diphosphate synthase (GDS) and limonene synthase (LS) in plastid.
  • TPGDSTPLS geranyl diphosphate synthase
  • GDS geranyl diphosphate synthase
  • LS limonene synthase
  • FIG. 26 Shows an exemplary E. coli cloning vector (pNaMluIOleosinGDS) with an oleosin promoter, GDS and an oleosin terminator.
  • pNaMluIOleosinGDS E. coli cloning vector
  • FIG. 27 Shows an exemplary E. coli cloning vector (pNaAscINapinLS) with a napin promoter, LS and a glycinin terminator.
  • FIG. 28 Shows an exemplary plant expression vector (GDSLS) for co-expressing geranyl diphosphate synthase (GDS) and limonene synthase (LS) in cytosol.
  • GDSLS geranyl diphosphate synthase
  • LS limonene synthase
  • FIG. 29 Shows an exemplary E. coli expression vector (pET28)
  • FIG. 30 Shows an exemplary E. coli expression vector (pET28-GDS9aaLS) for a fusion protein of geranyl diphosphate synthase (GDS) and limonene synthase (LS).
  • GDS geranyl diphosphate synthase
  • LS limonene synthase
  • FIG. 31 Shows an exemplary E. coli expression vector (pET28-LS9aaGDS) for a fusion protein of limonene synthase (LS) and geranyl diphosphate synthase (GDS).
  • pET28-LS9aaGDS E. coli expression vector for a fusion protein of limonene synthase (LS) and geranyl diphosphate synthase (GDS).
  • FIG. 32 Shows a graphical representation of terpene metabolic pathway directed to producing (4S)-limonene and 5-epi-aristolochene.
  • GDS and FDS are prenyltransferases;
  • LS and EAS are terpene synthases.
  • Peppermint (4S)-limonene is produced in plastid via the non-mevalonate pathway.
  • Tobacco 5-epi-aristolochene is produced in cytosol via the mevalonate pathway.
  • GDS geranyl diphosphate synthase; LS, (4S)-limonene synthase; FDS, farnesyl diphosphate synthase; EAS, 5-epi-aristolochene synthase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase with its own transit peptide; TP, Rubisco small subunit transit peptide; OP, oleosin promoter; NP, napin promoter; GP, glycinin promoter; OT, oleosin terminator; GT, glycinin terminator.
  • FIG. 33 Shows detection of (4S)-limonene and 5-epi-aristolochene in the transgenic camelina seeds. Diethyl ether extract from camelina seed was analyzed by GC-MS. Each extract was prepared from 10 mature seeds.
  • (4S)-Limonene constituted 97% of the total monoterpenes calculated from the signal intensities.
  • FIG. 34 Shows (4S)-limonene and 5-epi-aristolochene in the transgenic camelina seeds.
  • the (4S)-limonene content was calculated by GC-MS with standard (4S)-limonene.
  • the 5-epi-aristolochene content was calculated by GC-MS with standard valencene (analog of 5-epi-aristrolochene).
  • ND not detected; wt, wild-type plant. Data are means ⁇ SD from analysis of at least 3 independent seed batches containing 10 seeds.
  • FIG. 35 Shows confirmation of transgene expression in Example 2.
  • FIG. 36 Shows enzyme assay of the transgenes in Example 2.
  • the specific activities of GDS and LS were determined by GC-MS. Crude protein of the transgenic camelina mature seeds was incubated with substrates.
  • GDS reaction was started by adding DMAPP and IPP as substrates.
  • the reaction mixture contained LS recombinant protein (50 ⁇ g) purified from E. coli .
  • LS reaction was started with GPP as substrate. Enzymatically produced (4S)-limonene amount was quantified by GC-MS.
  • FIG. 37 Shows camelina transgenic lines expressing a fusion protein of GDS and LS.
  • the TPGDSLS fusion (plastid) binary vector contains a fusion protein comprised of a transit peptide (TP), GDS, a 9 amino acid linker and LS.
  • the TPLSGDS fusion (plastid) binary vector contains a fusion protein comprised of a transit peptide (TP), LS, a 9 amino acid linker and GDS.
  • the GDSLS fusion (cytosol) binary vector contains a fusion protein comprised of GDS, a 9 amino acid linker and LS.
  • the gene expression was controlled by the seed-specific oleosin promoter (OP). OT, oleosin terminator.
  • OP seed-specific oleosin promoter
  • FIG. 38 Shows (4S)-limonene in TPGDS TPLS plastid type T 4 and T 5 seeds.
  • the (4S)-limonene content was calculated from TPGDS TPLS (plastid) T 4 and T 5 seed (lines #11 and 29) by GC-MS with standard (4S)-limonene.
  • Data are means ⁇ SD from analysis of at least 3 independent seed batches containing 10 seeds.
  • the terms “cell,” “cells,” “cell line,” “host cell,” and “host cells,” are used interchangeably and, encompass animal cells and include plant, invertebrate, non-mammalian vertebrate, insect, algal, and mammalian cells. All such designations include cell populations and progeny.
  • the terms “transformants” and “transfectants” include the primary subject cell and cell lines derived therefrom without regard for the number of transfers.
  • “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag).
  • amino acid groups defined in this manner include: a “charged/polar group,” consisting of Glu, Asp, Asn, Gln, Lys, Arg and His; an “aromatic, or cyclic group,” consisting of Pro, Phe, Tyr and Trp; and an “aliphatic group” consisting of Gly, Ala, Val, Leu, Ile, Met, Ser, Thr and Cys.
  • subgroups can also be identified, for example, the group of charged/polar amino acids can be sub-divided into the sub-groups consisting of the “positively-charged sub-group,” consisting of Lys, Arg and His; the negatively-charged sub-group,” consisting of Glu and Asp, and the “polar sub-group” consisting of Asn and Gln.
  • the aromatic or cyclic group can be sub-divided into the sub-groups consisting of the “nitrogen ring sub-group,” consisting of Pro, His and Trp; and the “phenyl sub-group” consisting of Phe and Tyr.
  • the aliphatic group can be sub-divided into the sub-groups consisting of the “large aliphatic non-polar sub-group,” consisting of Val, Leu and Ile; the “aliphatic slightly-polar sub-group,” consisting of Met, Ser, Thr and Cys; and the “small-residue sub-group,” consisting of Gly and Ala.
  • conservative mutations include substitutions of amino acids within the sub-groups above, for example, Lys for Arg and vice versa such that a positive charge can be maintained; Glu for Asp and vice versa such that a negative charge can be maintained; Ser for Thr such that a free —OH can be maintained; and Gln for Asn such that a free —NH 2 can be maintained.
  • expression refers to transcription and/or translation of a nucleotide sequence within a host cell.
  • the level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired polypeptide encoded by the selected sequence.
  • mRNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA or by PCR.
  • Proteins encoded by a selected sequence can be quantified by various methods including, but not limited to, e.g., ELISA, Western blotting, radioimmunoassays, immunoprecipitation, assaying for the biological activity of the protein, or by immunostaining of the protein followed by FACS analysis.
  • “Expression control sequences” are regulatory sequences of nucleic acids, or the corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA, or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, and the like, that have the ability to affect the transcription or translation, or subcellular, or cellular location of a coding sequence in a host cell. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • a “gene” is a sequence of nucleotides which code for a functional gene product.
  • a gene product is a functional protein.
  • a gene product can also be another type of molecule in a cell, such as RNA (e.g., a tRNA or an rRNA).
  • a gene may also comprise expression control sequences (i.e., non-coding) sequences as well as coding sequences and introns.
  • the transcribed region of the gene may also include untranslated regions including introns, a 5′-untranslated region (5′-UTR) and a 3′-untranslated region (3′-UTR).
  • heterologous refers to a nucleic acid or protein which has been introduced into an organism (such as a plant, animal, or prokaryotic cell), or a nucleic acid molecule (such as chromosome, vector, or nucleic acid construct), which are derived from another source, or which are from the same source, but are located in a different (i.e. non native) context.
  • homologous refers to a nucleic acid or protein which is naturally occurring within an organism (such as a plant, animal, or prokaryotic cell) and is in its native context or location, or a nucleic acid molecule (such as chromosome, vector, or nucleic acid construct) which is derived from the same source, and which is in its native context.
  • homologous can also refer to the relationship between two proteins that possess a “common evolutionary origin”, including proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species of animal, as well as homologous proteins from different species of animal (for example, myosin light chain polypeptide, etc.; see Reeck et al., (1987) Cell, 50:667).
  • proteins and their encoding nucleic acids
  • sequence homology as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.
  • the term “homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs.
  • the nucleic acid and protein sequences of the present invention can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and BLAST
  • the term “increase” or the related terms “increased”, “enhance” or “enhanced” refers to a statistically significant increase.
  • the terms generally refer to at least a 10% increase in a given parameter, and can encompass at least a 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, 95% increase, 97% increase, 99% or even a 100% increase over the control value.
  • isolated when used to describe a protein or nucleic acid, means that the material has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with research, diagnostic or therapeutic uses for the protein or nucleic acid, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the protein or nucleic acid will be purified to at least 95% homogeneity as assessed by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated protein includes protein in situ within recombinant cells, since at least one component of the protein of interest's natural environment will not be present. Ordinarily, however, isolated proteins and nucleic acids will be prepared by at least one purification step.
  • identity means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; & Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always; 0) and N (penalty score for mismatching residues; always; 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the ⁇ 27 cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W. T. and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1, in another embodiment less than about 0.01, and in still another embodiment less than about 0.001.
  • terpene refers to any organic derived molecule formed directly from one or more isoprene, (C 5 H 8 ) units.
  • the term “hemiterpenes” refers to any cyclic or acyclic terpene containing one isoprene units.
  • the term “monoterpene” refers to any cyclic or acyclic terpene containing two isoprene units.
  • the term “sesquiterpene” refers to any cyclic or acyclic terpene containing three isoprene units.
  • Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers.
  • Exemplary terpenes are provided for example in CRC Handbook of Terpenoids: Acyclic, Monocyclic, Bicyclic, Tricyclic, and Tetracyclic Terpenoids (1989) by S. Dev. ISBN 9780849336119; HANDBOOK OF TERPENOIDS, VOLUME 1 by DEV S. and NAGASAMPAGI ISBN: 0849336112; Chapter 13. Terpenoids and steroids of Annu. Rep. Prog. Chem., Sect. B: Org. Chem., 1985, 82, 353-375 by J. R. Hanson and in Degenhardt et al., Phytochemistry (2009) 70 1621-1637, all of which are incorporated by reference in their entirety. Representative exemplary terpenes are provided by way of illustration, but not limitation, in FIG. 1 .
  • oil seed plant or “oil crop” refers to plants that produce seeds or fruit with a high oil content, e.g., greater than about 10%.
  • exemplary oil seed or oil crop plants include, for example, plants of the genus Camelina , coconut, cotton, peanut, rapeseed (canola), safflower, sesame, soybean, wheat, flax, sunflower, olive, corn, palm, sugarcane, castor bean, switchgrass, Miscanthus , and Jatropha.
  • a nucleic acid molecule according to the invention includes one or more DNA elements capable of opening chromatin and/or maintaining chromatin in an open state operably linked to a nucleotide sequence encoding a recombinant protein.
  • a nucleic acid molecule may additionally include one or more DNA or RNA nucleotide sequences chosen from: (a) a nucleotide sequence capable of increasing translation; (b) a nucleotide sequence capable of increasing secretion of the recombinant protein outside a cell; (c) a nucleotide sequence capable of increasing the mRNA stability, and (d) a nucleotide sequence capable of binding a trans-acting factor to modulate transcription or translation, where such nucleotide sequences are operatively linked to a nucleotide sequence encoding a recombinant protein.
  • nucleotide sequences that are operably linked are contiguous and, where necessary, in reading frame.
  • an operably linked DNA element capable of opening chromatin and/or maintaining chromatin in an open state is generally located upstream of a nucleotide sequence encoding a recombinant protein; it is not necessarily contiguous with it.
  • Operable linking of various nucleotide sequences is accomplished by recombinant methods well known in the art, e.g. using PCR methodology, by ligation at suitable restrictions sites or by annealing. Synthetic oligonucleotide linkers or adaptors can be used in accord with conventional practice if suitable restriction sites are not present.
  • plants that naturally produce monoterpenes refers to any plant, algae, or fungi that produces detectable levels of any terpene.
  • plants that naturally produce terpenes include for example, Pinus taeda , loblolly pine, Pinaceae, forest, Juniperus virginiana , cedar, Cupressaceae, tree, Magnolia grandiflora, magnolia , Magnoliaceae, flower and fruit, Umbellularia californica , California bay laurel, Lauraceae, branches with fruit, Cinnamomum camphora , camphor tree, Lauraceae, branch with flowers, Cananga odorata , ylang-ylang, Annonaceae, branch with flower, Citrus limon , lemon, Rutaceae, branch with fruits, Bursera gummifera , Burseraceae, tree, Rosa damascena , rose, Rosaceae, plant with flower, Pelargonium sp.,
  • nucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
  • a nucleic acid molecule can take many different forms, e.g., a gene or gene fragment, one or more exons, one or more introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches.
  • a polynucleotide includes not only naturally occurring bases such as A, T, U, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
  • a “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence.
  • the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease S1) can be found within a promoter sequence, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the ⁇ 10 and ⁇ 35 consensus sequences.
  • promoters including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art.
  • Representative sources include for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources.
  • Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3′ or 5′ direction).
  • Non-limiting examples of promoters active in plants include, for example nopaline synthase (nos) promoter and octopine synthase (ocs) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens and the caulimovirus promoters such as the Cauliflower Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No. 5,352,605), CaMV 35S promoter with a duplicated enhancer (U.S. Pat. Nos.
  • CaMV Cauliflower Mosaic Virus
  • purified refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell. Methods for purification are well-known in the art.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 75% pure, and more preferably still at least 95% pure.
  • Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • the term “substantially pure” indicates the highest degree of purity, which can be achieved using conventional purification techniques known in the art.
  • sequence similarity refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
  • sequence similarity when modified with an adverb such as “highly”, may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • two nucleic acid sequences are “substantially homologous” or “substantially similar” when at least about 85%, and more preferably at least about 90% or at least about 95% of the nucleotides match over a defined length of the nucleic acid sequences, as determined by a sequence comparison algorithm known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc.
  • BLAST Altschul et al.
  • FASTA DNA Strider
  • CLUSTAL etc.
  • An example of such a sequence is an allelic or species variant of the specific genes of the present invention.
  • Sequences that are substantially homologous may also be identified by hybridization, e.g., in a Southern hybridization experiment under, e.g., stringent conditions as defined for that particular system.
  • two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 90% of the amino acid residues are identical.
  • Two sequences are functionally identical when greater than about 95% of the amino acid residues are similar.
  • the similar or homologous polypeptide sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Version 7, Madison, Wis.) pileup program, or using any of the programs and algorithms described above.
  • transgenic plant is one whose genome has been altered by the incorporation of heterologous genetic material, e.g. by transformation as described herein.
  • the term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a transgenic plant, so long as the progeny contains the heterologous genetic material in its genome.
  • transformation refers to the transfer of one or more nucleic acid molecules into a host cell or organism.
  • Methods of introducing nucleic acid molecules into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, scrape loading, ballistic introduction, or infection with viruses or other infectious agents.
  • Transformed in the context of a cell, refers to a host cell or organism into which a recombinant or heterologous nucleic acid molecule (e.g., one or more DNA constructs or RNA, or siRNA counterparts) has been introduced.
  • the nucleic acid molecule can be stably expressed (i.e. maintained in a functional form in the cell for longer than about three months) or non-stably maintained in a functional form in the cell for less than three months i.e. is transiently expressed.
  • “transformed,” “transformant,” and “transgenic” cells have been through the transformation process and contain foreign nucleic acid.
  • the term “untransformed” refers to cells that have not been through the transformation process.
  • the present invention includes methods, DNA constructs, and transgenic plants that exhibit enhanced rates of terpene production and improved terpene content.
  • such methods and transgenic plants are created through the over expression of fusion proteins comprising either the GPP synthase large and small subunits, and limonene synthase, or one or more of these subunits fused to limonene synthase.
  • the enzymes are expressed with plastids of seed tissues.
  • the current invention includes a method for the production of a terpene, comprising the steps of:
  • fusion protein and limonene synthase are expressed primarily in the plant cell plastids.
  • the invention includes a method for the production of a monoterpene, comprising the step of:
  • fusion protein is expressed primarily in the plant cell plastids.
  • the fusion protein is expressed primarily in the seeds of the plant.
  • the terms “geranyl diphosphate synthase” or “GDP synthase” or “GDS” refers to all naturally-occurring and synthetic genes encoding a geranyl diphosphate synthase large or small subunit.
  • the geranyl diphosphate synthase is from a plant. In one aspect the geranyl diphosphate synthase is from plant that naturally produces terpenes.
  • polynucleotides can encode the geranyl diphosphate synthases of the invention.
  • polynucleotide sequence can be manipulated for various reasons. Examples include, but are not limited to, the incorporation of preferred codons to enhance the expression of the polynucleotide in various organisms (see generally Nakamura et al., Nuc. Acid. Res. (2000) 28 (1): 292).
  • silent mutations can be incorporated in order to introduce, or eliminate restriction sites, remove cryptic splice sites, or manipulate the ability of single stranded sequences to form stem-loop structures: (see, e.g., Zuker M., Nucl. Acid Res. (2003); 31(13): 3406-3415).
  • expression can be further optimized by including consensus sequences at and around the start codon.
  • Such codon optimization can be completed by standard analysis of the preferred codon usage for the host organism in question, and the synthesis of an optimized nucleic acid via standard DNA synthesis.
  • a number of companies provide such services on a fee for services basis and include for example, DNA2.0, (CA, USA) and Operon Technologies. (CA, USA).
  • the geranyl diphosphate synthase subunits may be in their native form, i.e., as different apo forms, or allelic variants as they appear in nature, which may differ in their amino acid sequence, for example, by proteolytic processing, including by truncation (e.g., from the N- or C-terminus or both) or other amino acid deletions, additions, insertions, substitutions.
  • Naturally-occurring chemical modifications including post-translational modifications and degradation products of the geranyl diphosphate synthase subunits are also specifically included in any of the methods of the invention including for example, pyroglutamyl, iso-aspartyl, proteolytic, phosphorylated, glycosylated, reduced, oxidatized, isomerized, and deaminated variants of the geranyl diphosphate synthase.
  • the geranyl diphosphate synthase subunits which may be used in any of the methods, DNA constructs, and plants of the invention may have amino acid sequences which are substantially homologous, or substantially similar to any of the native geranyl diphosphate synthase sequences, for example, to any of the native geranyl diphosphate synthase gene sequences listed in Tables D1 and D2.
  • the geranyl diphosphate synthase may have an amino acid sequence having at least 30% preferably at least 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identity with a geranyl diphosphate synthase listed in Tables D1 or D2.
  • the geranyl diphosphate synthase small subunit for use in any of the methods and plants of the present invention is at least 80% identical to the mature geranyl diphosphate synthase (shown without the native transit peptide below) small subunit from Mentha ⁇ piperita :
  • the geranyl diphosphate synthase large subunit for use in any of the methods and plants of the present invention is at least 80% identical to the mature geranyl diphosphate synthase (shown without the native transit peptide below) large subunit from Mentha ⁇ piperita :
  • the geranyl diphosphate synthase subunits and fusion proteins thereof can include modified forms in which the native transit peptide has been removed, or replaced with another synthetic, or naturally occurring, transit peptide sequence.
  • Transit sequences are joined to the coding sequence of an expressed gene, and are removed post-translationally from the initial translation product.
  • Various transit peptides which function as described herein are well known in the art, and are described in, for example, Johnson et al. The Plant Cell (1990) 2:525-532; Sauer et al. EMBO J. (1990) 9:3045-3050; Mueckler et al. Science (1985) 229:941-945; Von Heijne, Eur. J. Biochem. (1983) 133:17-21; Yon Heijne, J. Mol. Biol. (1986) 189:239-242; Iturriaga et al.
  • the transit peptide may comprise the pea RuBisCO small subunit transit peptide:
  • the term “limonene synthase”, or “LS” refers to all naturally-occurring and synthetic genes encoding a limonene synthase.
  • limonene synthases may exist in two forms; The (S) or ( ⁇ ) forms producing the ( ⁇ )-(4S)-limonene enantiomer and the (R) or (+) forms producing the (+)-(4R)-limonene enantiomer.
  • the limonene synthase is from a plant.
  • the limonene synthase is from plant that naturally produces terpenes.
  • the limonene synthase is the ( ⁇ ) or (S) form.
  • the limonene synthase is the (+) or (R) form.
  • polynucleotide sequence can be manipulated for various reasons. Examples include, but are not limited to, the incorporation of preferred codons to enhance the expression of the polynucleotide in various organisms (see generally Nakamura et al., Nuc. Acid. Res. (2000) 28 (1): 292).
  • silent mutations can be incorporated in order to introduce, or eliminate restriction sites, remove cryptic splice sites, or manipulate the ability of single stranded sequences to form stem-loop structures: (see, e.g., Zuker M., Nucl. Acid Res. (2003); 31(13): 3406-3415).
  • expression can be further optimized by including consensus sequences at and around the start codon.
  • Such codon optimization can be completed by standard analysis of the preferred codon usage for the host organism in question, and the synthesis of an optimized nucleic acid via standard DNA synthesis.
  • a number of companies provide such services on a fee for services basis and include for example, DNA2.0, (CA, USA) and Operon Technologies. (CA, USA).
  • the limonene synthase may be in its native form, i.e., as different apo forms, or allelic variants as they appear in nature, which may differ in their amino acid sequence, for example, by proteolytic processing, including by truncation (e.g., from the N- or C-terminus or both) or other amino acid deletions, additions, insertions, substitutions.
  • Naturally-occurring chemical modifications including post-translational modifications and degradation products of the limonene synthase are also specifically included in any of the methods of the invention including for example, pyroglutamyl, iso-aspartyl, proteolytic, phosphorylated, glycosylated, reduced, oxidatized, isomerized, and deaminated variants of the limonene synthase.
  • the limonene synthase which may be used in any of the methods, fusion proteins, DNA constructs, and plants of the invention may have amino acid sequences which are substantially homologous, or substantially similar to any of the native limonene synthase sequences, for example, to any of the native limonene synthase gene sequences listed in Table D3.
  • the limonene synthase may have an amino acid sequence having at least 30% preferably at least 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identity with a limonene synthase listed in Table D3.
  • the limonene synthase for use in any of the methods and plants of the present invention is at least 80% identical to the mature limonene synthase from Mentha ⁇ piperita (shown both with and without the native transit peptide):
  • the limonene synthase, and fusion proteins thereof can include modified forms in which the native transit peptide has been removed, or replaced with another synthetic, or naturally occurring, transit peptide sequence derived from another well characterized chloroplast localized proteins. Such transit sequences are joined to the coding sequence of an expressed gene, and are removed post-translationally from the initial translation product.
  • Various transit peptides which function as described herein are well known in the art, and are described in, for example, Johnson et al. The Plant Cell (1990) 2:525-532; Sauer et al. EMBO J. (1990) 9:3045-3050; Mueckler et al.
  • the transit peptide may comprise the pea RuBisCO small subunit transit peptide:
  • the invention includes the further expression, or introduction of further synthetic enzymes to promote the formation of distinct classes of terpenes.
  • Representative exemplary enzymes include for example, the enzymes listed in Table D4.
  • the invention includes fusion proteins of either the GDP synthase large and small subunits, or one or more of these subunits fused to limonene synthase. In certain embodiments these fusion proteins may increase the relative enzymatic specific activity and/or efficiency of terpene synthesis.
  • fusion proteins include, i) the fusion of the GDP synthase large subunit to the GDP synthase small subunit; ii) the fusion GDP synthase large subunit to limonene synthase; iii) the fusion of the GDP synthase small subunit to limonene synthase. It will be appreciated that any of such fusion proteins can be arranged in a number of different of relative orientations. Specific embodiments contemplated herein include:
  • a flexible molecular linker optionally may be interposed between, and covalently join, any of the transit peptides, GPP synthase subunits and limonene synthases disclosed herein. Any such fusion protein may be used in any of the methods, proteins, polynucleotides and host cells of the present invention.
  • the construct shown includes a 10 amino acid linker (SSNNLGIEGR (SEQ ID NO:34)), with the native transit peptide sequences removed from the GDS large and small subunits, and with a 5′ transit peptide from the pea RuBisCO small subunit.
  • the construct shown includes a 9 amino acid linker (SGGSGGSGG (SEQ ID NO:36)), linking the limonene synthase to the GDS (small subunit), with the native transit peptide sequences removed from the GDS subunit and limonene synthase, and with the transit peptide from the pea RuBisCO small subunit added to the N-terminus of limonene synthase.
  • SGGSGGSGG SEQ ID NO:36
  • the construct shown also includes a 9 amino acid linker (SGGSGGSGG (SEQ ID NO:36)) linking GDS (large) to limonene synthase.
  • SGGSGGSGG SEQ ID NO:36
  • the native transit peptide sequences have been removed from the GDS large and small subunits, and limonene synthase, and the 5′ transit peptide from the pea RuBisCO small subunit added to the N-terminus.
  • the DNA constructs, and expression vectors of the invention include separate expression vectors each including either the isolated geranyl diphosphate synthase or limonene synthase, or the previously described fusion proteins thereof.
  • the DNA constructs and expression vectors for the GDS and limonene fusion proteins comprise polynucleotide sequences encoding any of the previously described fusion proteins operatively coupled to a promoter, transit peptide sequence and transcriptional terminator for efficient expression in the organism of interest.
  • the geranyl diphosphate synthase is codon optimized for expression in the organism of interest.
  • the geranyl diphosphate synthase DNA constructs and expression vectors of the invention further comprise polynucleotide sequences encoding one or more of the following elements i) a selectable marker gene to enable antibiotic selection, ii) a screenable marker gene to enable visual identification of transformed cells, and iii) T-element DNA sequences to enable Agrobacterium tumefaciens mediated transformation.
  • exemplary expression cassettes are described in the Examples.
  • the DNA constructs and expression vectors for the limonene synthase comprise polynucleotide sequences encoding any of the previously described limonene synthase, genes (Table D2) operatively coupled to a promoter, and transcriptional terminator for efficient expression in the organism of interest.
  • the limonene synthase is codon optimized for expression in the photosynthetic organism of interest.
  • the limonene synthase gene encodes a limonene synthase of Mentha ⁇ piperita.
  • the limonene synthase DNA constructs and expression vectors of the invention further comprise polynucleotide sequences encoding one or more of the following elements i) a selectable marker gene to enable antibiotic selection, ii) a screenable marker gene to enable visual identification of transformed cells, and iii) T-element DNA sequences to enable Agrobacterium tumefaciens mediated transformation.
  • exemplary expression cassettes are described in the Examples.
  • the DNA constructs, and expression vectors of the invention include expression vectors comprising nucleic acid sequences encoding i) the GDS large and small subunit fusion protein and ii) a limonene synthase gene.
  • Exemplary expression cassettes are described in the Examples.
  • expression cassettes represents only illustrative examples of expression cassettes that could be readily constructed, and is not intended to represent an exhaustive list of all possible DNA constructs or expression cassettes that could be constructed.
  • expression vectors suitable for use in expressing the claimed DNA constructs in plants, and methods for their construction are generally well known, and need not be limited. These techniques, including techniques for nucleic acid manipulation of genes such as subcloning a subject promoter, or nucleic acid sequences encoding a gene of interest into expression vectors, labeling probes, DNA hybridization, and the like, and are described generally in Sambrook, et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference.
  • DNA constructs comprising an expression cassette for the gene of interest can then be inserted into a variety of expression vectors.
  • Such vectors include expression vectors that are useful in the transformation of plant cells. Many other such vectors useful in the transformation of plant cells can be constructed by the use of recombinant DNA techniques well known to those of skill in the art as described above.
  • Exemplary expression vectors for expression in protoplasts or plant tissues include pUC 18/19 or pUC 118/119 (GIBCO BRL, Inc., MD); pBluescript SK (+/ ⁇ ) and pBluescript KS (+/ ⁇ ) (STRATAGENE, La Jolla, Calif.); pT7Blue T-vector (NOVAGEN, Inc., WI); pGEM-3Z/4Z (PROMEGA Inc., Madison, Wis.), and the like vectors, such as is described herein
  • Exemplary vectors for expression using Agrobacterium tumefaciens -mediated plant transformation include for example, pBin 19 (CLONETECH), Frisch et al, Plant Mol.
  • DNA constructs will typically include expression control sequences comprising promoters to drive expression of the limonene synthase and geranyl diphosphate synthase within the plastids of the photosynthetic organism. Promoters may provide ubiquitous, cell type specific, constitutive promoter or inducible promoter expression. Basal promoters in plants typically comprise canonical regions associated with the initiation of transcription, such as CAAT and TATA boxes.
  • the TATA box element is usually located approximately 20 to 35 nucleotides upstream of the initiation site of transcription.
  • the CAAT box element is usually located approximately 40 to 200 nucleotides upstream of the start site of transcription.
  • basal promoter elements result in the synthesis of an RNA transcript comprising nucleotides upstream of the translational ATG start site.
  • the region of RNA upstream of the ATG is commonly referred to as a 5′ untranslated region or 5′ UTR.
  • basal promoters may be altered to contain “enhancer DNA” to assist in elevating gene expression.
  • certain DNA elements can be used to enhance the transcription of DNA.
  • enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted upstream (5′) or downstream (3′) to the coding sequence.
  • these 5′ enhancer DNA elements are introns.
  • the introns that are particularly useful as enhancer DNA are the 5′ introns from the rice actin 1 gene (see U.S. Pat. No. 5,641,876), the rice actin 2 gene, the maize alcohol dehydrogenase gene, the maize heat shock protein 70 gene (U.S. Pat. No. 5,593,874), the maize shrunken 1 gene, the light sensitive 1 gene of Solanum tuberosum , and the heat shock protein 70 gene of Petunia hybrida (U.S. Pat. No. 5,659,122).
  • promoter selection can be based on expression profile and expression level.
  • the following are representative non-limiting examples of promoters that can be used in the expression cassettes.
  • Constitutive promoters typically provide for the constant and substantially uniform production of proteins in all tissues.
  • Exemplary constitutive promoters include for example, the core promoter of the Rsyn7 (U.S. patent application Ser. No. 08/661,601), the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.
  • Tissue-specific promoters include those described in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
  • Root specific promoters include, for example, those disclosed in Hire, et al (1992) Plant Mol. Biology, 20(2): 207-218; Keller and Baumgartner, (1991) The Plant Cell, 3(10): 1051-1061; Sanger et al. (1990) Plant Mol.
  • Seed-preferred promoters includes both seed-specific promoters (those promoters active during seed development) as well as seed-germinating promoters (those promoters active during seed germination).
  • Such promoters include Cim1 (cytokinin-induced message); cZ19B1 (maize 19 KDa zein); milps (myo-inositol-1-phosphate synthase); celA (cellulose synthase); end1 (Hordeum verlgase mRNA clone END1); and imp3 (myo-inositol monophosphate-3).
  • particular promoters include phaseolin, napin, ⁇ -conglycinin, soybean lectin, and the like.
  • particular promoters include maize 15 Kd zein, 22 KD zein, 27 kD zein, waxy, shrnmken 1, shrunken 2, globulin 1, etc.
  • the DNA constructs, transgenic plants and methods use the oleosin promoter and/or napin promoter.
  • the double 35S promoter in pCGN1761ENX can be replaced with any other promoter of choice that will result in suitably high expression levels.
  • one of the chemically regulatable promoters described in U.S. Pat. Nos. 5,614,395 and 5,880,333 can replace the double 35S promoter.
  • the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites.
  • the selected target gene coding sequence can be inserted into this vector, and the fusion products (i.e., promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described below.
  • Various chemical regulators can be employed to induce expression of the selected coding sequence in the plants transformed according to the presently disclosed subject matter, including the benzothiadiazole, isonicotinic acid, salicylic acid and Ecdysone receptor ligands compounds disclosed in U.S. Pat. Nos. 5,523,311, 5,614,395, and 5,880,333 herein incorporated by reference.
  • transcriptional terminators are available for use in the DNA constructs of the invention. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation.
  • RNA polymerase III terminators are those that are known to function in the relevant plant system.
  • Representative plant transcriptional terminators include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator (NOS ter), and the pea rbcS E9 terminator.
  • the inventions utilize the oleosin terminator and/or napin terminator.
  • these terminators typically comprise a ⁇ 52 run of 5 or more consecutive thymidine residues.
  • an RNA polymerase III terminator comprises the sequence TTTTTTT. These can be used in both monocotyledons and dicotyledons.
  • Transit peptides can be identified in the primary amino acid sequences of the preproteins by those ordinarily skilled in the art. For example, see Colby et al. (1993) J. Biol. Chem. 268(31):23016-23024, for the transit peptide sequence of limonene synthase. In certain embodiments, the transit peptide sequence form the RuBisCO small subunit transit peptide is used.
  • nucleic acids of the presently disclosed subject matter Numerous sequences have been found to enhance the expression of an operatively lined nucleic acid sequence, and these sequences can be used in conjunction with the nucleic acids of the presently disclosed subject matter to increase their expression in transgenic plants.
  • intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
  • the introns of the maize Adbl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells.
  • Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene.
  • the intron from the maize bronzes gene had a similar effect in enhancing expression.
  • Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
  • leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMY) have been shown to be effective in enhancing expression.
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AY Alfalfa Mosaic Virus
  • selection markers can be included in the DNA constructs of the invention.
  • Selection markers used routinely in transformation include the npt II gene (Kan), which confers resistance to kanamycin and related antibiotics, the bar gene, which confers resistance to the herbicide phosphinothricin, the hph gene, which confers resistance to the antibiotic hygromycin, the dhfr gene, which confers resistance to methotrexate, and the EPSP synthase gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642).
  • Screenable markers may also be employed in the DNA constructs of the present invention, including for example the ⁇ -glucuronidase or uidA gene (the protein product is commonly referred to as GUS), isolated from E. coli , which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a ⁇ -lactamase gene, which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene, which encodes a catechol dioxygenase that can convert chromogenic catechols; an ⁇ -amylase gene; a tyrosinase gene which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily-detectable compound melanin;
  • the R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue.
  • Maize strains can have one, or as many as four, R alleles which combine to regulate pigmentation in a developmental and tissue specific manner.
  • an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector.
  • a maize line carries dominant alleles for genes encoding for the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation.
  • Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR112, a K55 derivative which has the genotype r-g, b, Pl.
  • Wisconsin 22 which contains the rg-Stadler allele and TR112
  • TR112 a K55 derivative which has the genotype r-g, b, Pl.
  • any genotype of maize can be utilized if the Cl and R alleles are introduced together.
  • screenable markers provide for visible light emission or fluorescence as a screenable phenotype.
  • Suitable screenable markers contemplated for use in the present invention include firefly luciferase, encoded by the lux gene.
  • the presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It also is envisioned that this system may be developed for population screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
  • the DNA constructs of the present invention typically contain a marker gene which confers a selectable phenotype on the plant cells.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorsulfuron or Basta.
  • antibiotic resistance such as resistance to kanamycin, G418, bleomycin, hygromycin
  • herbicide resistance such as resistance to chlorsulfuron or Basta.
  • DNA constructs can be introduced into the genome of the desired plant host by a variety of conventional techniques.
  • the DNA construct may be introduced directly into the DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts.
  • the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA micro-particle bombardment.
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Microinjection techniques are known in the art and well described in the scientific and patent literature.
  • the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al, (1984) EMBO J., 3:2717-2722.
  • Electroporation techniques are described in Fromm et al, (1985) Proc. Natl. Acad. Sci. USA, 82:5824.
  • Biolistic transformation techniques are described in Klein et al, (1987) Nature 327:70-7. The full disclosures of all references cited are incorporated herein by reference.
  • a variation involves high velocity biolistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al, (1987) Nature, 327:70-73,). Although typically only a single introduction of a new nucleic acid segment is required, this method particularly provides for multiple introductions.
  • Agrobacterium tumefaciens -meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al, (1984) Science, 233:496-498, and Fraley et al, (1983) Proc. Natl. Acad. Sci. USA, 90:4803.
  • a plant cell, an explant, a meristem or a seed is infected with Agrobacterium tumefaciens transformed with the segment.
  • the transformed plant cells are grown to form shoots, roots, and develop further into plants.
  • the nucleic acid segments can be introduced into appropriate plant cells, for example, by means of the Ti plasmid of Agrobacterium tumefaciens .
  • the Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens , and is stably integrated into the plant genome (Horsch et al, (1984) Science, 233:496-498; Fraley et al, (1983) Proc. Nat'l. Acad. Sci. U.S.A., 80:4803.
  • Ti plasmids contain two regions essential for the production of transformed cells. One of these, named transfer DNA (T DNA), induces tumor formation. The other, termed virulent region, is essential for the introduction of the T DNA into plants.
  • T DNA transfer DNA
  • the transfer DNA region which transfers to the plant genome, can be increased in size by the insertion of the foreign nucleic acid sequence without its transferring ability being affected. By removing the tumor-causing genes so that they no longer interfere, the modified Ti plasmid can then be used as a vector for the transfer of the gene constructs of the invention into an appropriate plant cell, such being a “disabled Ti vector”.
  • All plant cells which can be transformed by Agrobacterium and whole plants regenerated from the transformed cells can also be transformed according to the invention so as to produce transformed whole plants which contain the transferred foreign nucleic acid sequence.
  • There are various ways to transform plant cells with Agrobacterium including: (1) co-cultivation of Agrobacterium with cultured isolated protoplasts, (2) co-cultivation of cells or tissues with Agrobacterium , or (3) transformation of seeds, apices or meristems with Agrobacterium .
  • Method (1) requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
  • Method (2) requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
  • Method (3) requires micropropagation.
  • T-DNA containing plasmid In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid. Any one of a number of T-DNA containing plasmids can be used, the only requirement is that one be able to select independently for each of the two plasmids.
  • those plant cells or plants transformed by the Ti plasmid so that the desired DNA segment is integrated can be selected by an appropriate phenotypic marker.
  • phenotypic markers include, but are not limited to, antibiotic resistance, herbicide resistance or visual observation. Other phenotypic markers are known in the art and may be used in this invention.
  • the present invention embraces use of the claimed DNA constructs in transformation of any plant, including both dicots and monocots. Transformation of dicots is described in references above. Transformation of monocots is known using various techniques including electroporation (e.g., Shimamoto et al, (1992) Nature, 338:274-276; ballistics (e.g., European Patent Application 270,356); and Agrobacterium (e.g., Bytebier et al, (1987) Proc. Nat'l Acad. Sci. USA, 84:5345-5349).
  • electroporation e.g., Shimamoto et al, (1992) Nature, 338:274-276
  • ballistics e.g., European Patent Application 270,356
  • Agrobacterium e.g., Bytebier et al, (1987) Proc. Nat'l Acad. Sci. USA, 84:5345-5349.
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the desired transformed phenotype.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium typically relying on a biocide and/or herbicide marker which has been introduced together with the nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al, Handbook of Plant Cell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof.
  • DNA is introduced into only a small percentage of target cells in any one experiment.
  • a means for selecting those cells that are stably transformed is to introduce into the host cell, a marker gene which confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide.
  • antibiotics which may be used include the aminoglycoside antibiotics neomycin, kanamycin, G418 and paromomycin, or the antibiotic hygromycin.
  • aminoglycoside antibiotics Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphostransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.
  • aminoglycoside phosphostransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I
  • hygromycin phosphotransferase Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphostransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I
  • NPT II neomycin phosphotransferase II
  • hygromycin phosphotransferase Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphostransferase
  • Potentially transformed cells then are exposed to the selective agent.
  • population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival.
  • Cells may be tested further to confirm stable integration of the exogenous DNA. Using the techniques disclosed herein, greater than 40% of bombarded embryos may yield transformants.
  • Glyphosate inhibits the action of the enzyme EPSPS, which is active in the aromatic amino acid biosynthetic pathway. Inhibition of this enzyme leads to starvation for the amino acids phenylalanine, tyrosine, and tryptophan and secondary metabolites derived thereof.
  • EPSPS enzyme-activated glutathione
  • U.S. Pat. No. 4,535,060 describes the isolation of EPSPS mutations which confer glyphosate resistance on the Salmonella typhimurium gene for EPSPS, aroA.
  • the EPSPS gene was cloned from Zea mays and mutations similar to those found in a glyphosate resistant aroA gene were introduced in vitro. Mutant genes encoding glyphosate resistant EPSPS enzymes are described in, for example, PCT Publication WO 97/04103. The best characterized mutant EPSPS gene conferring glyphosate resistance comprises amino acid changes at residues 102 and 106, although it is anticipated that other mutations will also be useful (PCT Publication WO 97/04103). Furthermore, a naturally occurring glyphosate resistant EPSPS may be used, e.g., the CP4 gene isolated from Agrobacterium encodes a glyphosate resistant EPSPS (U.S. Pat. No. 5,627,061).
  • tissue is cultured for 0-28 days on nonselective medium and subsequently transferred to medium containing from 1-3 mg/l bialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or 1-3 mM glyphosate will typically be preferred, it is believed that ranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will find utility in the practice of the invention. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.
  • Bialaphos is a tripeptide antibiotic produced by Streptomyces hygroscopicus and is composed of phosphinothricin (PPT), an analogue of L-glutamic acid, and two L-alanine residues. Upon removal of the L-alanine residues by intracellular peptidases, the PPT is released and is a potent inhibitor of glutamine synthase (GS), a pivotal enzyme involved in ammonia assimilation and nitrogen metabolism. Synthetic PPT, the active ingredient in the herbicide LibertyTM also is effective as a selection agent. Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of the plant cells.
  • the organism producing bialaphos and other species of the genus Streptomyces also synthesizes an enzyme phosphinothricin acetyl transferase (PAT) which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogenes .
  • PAT phosphinothricin acetyl transferase
  • the use of the herbicide resistance gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE 3642 829 A, wherein the gene is isolated from Streptomyces viridochromogenes . In the bacterial source organism, this enzyme acetylates the free amino group of PPT preventing auto-toxicity.
  • the bar gene has been cloned and expressed in transgenic tobacco, tomato, potato, Brassica and maize (U.S. Pat. No. 5,550,318). In previous reports, some transgenic plants which expressed the resistance gene were completely resistant to commercial formulations of PPT and bialaphos in greenhouses.
  • the herbicide dalapon 2,2-dichloropropionic acid
  • the enzyme 2,2-dichloropropionic acid dehalogenase inactivates the herbicidal activity of 2,2-dichloropropionic acid and therefore confers herbicidal resistance on cells or plants expressing a gene encoding the dehalogenase enzyme (U.S. Pat. No. 5,780,708).
  • anthranilate synthase which confers resistance to certain amino acid analogs, e.g., 5-methyltryptophan or 6-methyl anthranilate, may be useful as a selectable marker gene.
  • an anthranilate synthase gene as a selectable marker was described in U.S. Pat. No. 5,508,468 and U.S. Pat. No. 6,118,047.
  • a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media. In a similar fashion, the introduction of the C1 and B genes will result in pigmented cells and/or tissues.
  • the enzyme luciferase may be used as a screenable marker in the context of the present invention.
  • cells expressing luciferase emit light which can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification.
  • the photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells that are expressing luciferase and manipulate cells expressing in real time.
  • Another screenable marker which may be used in a similar fashion is the gene coding for green fluorescent protein (GFP) or a gene coding for other fluorescing proteins such as DSRED® (Clontech, Palo Alto, Calif.).
  • a selection agent such as bialaphos or glyphosate
  • selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those that cause 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase or GFP would allow one to recover transformants from cell or tissue types that are not amenable to selection alone.
  • combinations of selection and screening may enable one to identify transformants in a wider variety of cell and tissue types. This may be efficiently achieved using a gene fusion between a selectable marker gene and a screenable marker gene, for example, between an NPTII gene and a GFP gene (WO 99/60129).
  • Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay may be cultured in media that supports regeneration of plants.
  • MS and N6 media may be modified by including further substances such as growth regulators.
  • Preferred growth regulators for plant regeneration include cytokines such as 6-benzylamino pelerine, peahen or the like, and abscise acid.
  • Media improvement in these and like ways has been found to facilitate the growth of cells at specific developmental stages.
  • Tissue may be maintained on a basic media with axing type growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, then transferred to media conducive to maturation of embroils. Cultures are transferred every 1-4 weeks, preferably every 2-3 weeks on this medium. Shoot development will signal the time to transfer to medium lacking growth regulators.
  • the transformed cells identified by selection or screening and cultured in an appropriate medium that supports regeneration, will then be allowed to mature into plants.
  • Developing plantlets were transferred to soilless plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 pap CO 2 , and 25-250 microeinsteins m ⁇ 2 s ⁇ 1 of light, prior to transfer to a greenhouse or growth chamber for maturation.
  • Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue.
  • cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Cons.
  • Regenerating plants are preferably grown at about 19 to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
  • Progeny may be recovered from transformed plants and tested for expression of the exogenous expressible gene.
  • seeds on transformed plants may occasionally require embryo rescue due to cessation of seed development and premature senescence of plants.
  • embryo rescue To rescue developing embryos, they are excised from surface-disinfected seeds 10-20 days post-pollination and cultured.
  • An embodiment of media used for culture at this stage comprises MS salts, 2% sucrose, and 5.5 g/l agarose.
  • embryo rescue large embryos (defined as greater than 3 mm in length) are germinated directly on an appropriate media. Embryos smaller than that may be cultured for 1 wk on media containing the above ingredients along with 10 ⁇ 5 M abscisic acid and then transferred to growth regulator-free medium for germination.
  • assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • Genomic DNA may be isolated from callus cell lines or any plant parts to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note, that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.
  • the presence of DNA elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant, but does not necessarily prove integration of the introduced gene into the host cell genome. Typically, DNA has been integrated into the genome of all transformants that demonstrate the presence of the gene through PCR analysis.
  • Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition, it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.
  • RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
  • PCR techniques referred to as RT-PCR, also may be used for detection and quantification of RNA produced from introduced genes.
  • RT-PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA.
  • PC techniques while useful, will not demonstrate integrity of the RNA product.
  • Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species also can be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.
  • TAQMAN® technology (Applied Biosystems, Foster City, Calif.) may be used to quantitate both DNA and RNA in a transgenic cell.
  • Southern blotting and PCR may be used to detect the gene(s) in question, they do not provide information as to whether the gene is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.
  • Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins.
  • Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
  • the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
  • Assay procedures also may be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and 14 C-acetyl CoA or for anthranilate synthase activity by following an increase in fluorescence as anthranilate is produced, to name two.
  • bioassays Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms, including but not limited to, analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins which change amino acid composition and may be detected by amino acid analysis, or by enzymes which change starch quantity which may be analyzed by near infrared reflectance spectrometry. Morphological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.
  • Southern blotting, PCR and RT-PCR techniques can be used to identify the presence or absence of a given transgene but, depending upon experimental design, may not specifically and uniquely identify identical or related transgene constructs located at different insertion points within the recipient genome.
  • To more precisely characterize the presence of transgenic material in a transformed plant one skilled in the art could identify the point of insertion of the transgene and, using the sequence of the recipient genome flanking the transgene, develop an assay that specifically and uniquely identifies a particular insertion event. Many methods can be used to determine the point of insertion such as, but not limited to, Genome WalkerTM technology (CLONTECH, Palo Alto, Calif.), VectoretteTM technology (Sigma, St.
  • restriction site oligonucleotide PCR uneven PCR (Chen and Wu, 1997) and generation of genomic DNA clones containing the transgene of interest in a vector such as, but not limited to, lambda phage.
  • two oligonucleotide primers can be designed, one wholly contained within the transgene and one wholly contained within the flanking sequence, which can be used together with the PCR technique to generate a PCR product unique to the inserted transgene.
  • the two oligonucleotide primers for use in PCR could be designed such that one primer is complementary to sequences in both the transgene and adjacent flanking sequence such that the primer spans the junction of the insertion site while the second primer could be homologous to sequences contained wholly within the transgene.
  • the two oligonucleotide primers for use in PCR could be designed such that one primer is complementary to sequences in both the transgene and adjacent flanking sequence such that the primer spans the junction of the insertion site while the second primer could be homologous to sequences contained wholly within the genomic sequence adjacent to the insertion site.
  • Confirmation of the PCR reaction may be monitored by, but not limited to, size analysis on gel electrophoresis, sequence analysis, hybridization of the PCR product to a specific radiolabeled DNA or RNA probe or to a molecular beacon, or use of the primers in conjugation with a TAQMAN probe and technology (Applied Biosystems, Foster City, Calif.).
  • site-specific integration or excision of transformation constructs prepared in accordance with the instant invention.
  • An advantage of site-specific integration or excision is that it can be used to overcome problems associated with conventional transformation techniques, in which transformation constructs typically randomly integrate into a host genome and multiple copies of a construct may integrate. This random insertion of introduced DNA into the genome of host cells can be detrimental to the cell if the foreign DNA inserts into an essential gene.
  • the expression of a transgene may be influenced by “position effects” caused by the surrounding genomic DNA.
  • Homologous recombination is a reaction between any pair of DNA sequences having a similar sequence of nucleotides, where the two sequences interact (recombine) to form a new recombinant DNA species.
  • the frequency of homologous recombination increases as the length of the shared nucleotide DNA sequences increases, and is higher with linearized plasmid molecules than with circularized plasmid molecules. Homologous recombination can occur between two DNA sequences that are less than identical, but the recombination frequency declines as the divergence between the two sequences increases.
  • Introduced DNA sequences can be targeted via homologous recombination by linking a DNA molecule of interest to sequences sharing homology with endogenous sequences of the host cell. Once the DNA enters the cell, the two homologous sequences can interact to insert the introduced DNA at the site where the homologous genomic DNA sequences were located. Therefore, the choice of homologous sequences contained on the introduced DNA will determine the site where the introduced DNA is integrated via homologous recombination. For example, if the DNA sequence of interest is linked to DNA sequences sharing homology to a single copy gene of a host plant cell, the DNA sequence of interest will be inserted via homologous recombination at only that single specific site.
  • the DNA sequence of interest is linked to DNA sequences sharing homology to a multicopy gene of the host eukaryotic cell, then the DNA sequence of interest can be inserted via homologous recombination at each of the specific sites where a copy of the gene is located.
  • DNA can be inserted into the host genome by a homologous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events).
  • a homologous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events).
  • a homologous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events).
  • the introduced DNA should contain sequences homologous to the selected gene.
  • a double recombination event can be achieved by flanking each end of the DNA sequence of interest (the sequence intended to be inserted into the genome) with DNA sequences homologous to the selected gene.
  • a homologous recombination event involving each of the homologous flanking regions will result in the insertion of the foreign DNA.
  • only those DNA sequences located between the two regions sharing genomic homology become integrated into the genome.
  • homologous recombination is a relatively rare event compared to random insertion events.
  • random integration of transgenes is more common in plants.
  • randomly inserted DNA sequences can be removed.
  • One manner of removing these random insertions is to utilize a site-specific recombinase system (U.S. Pat. No. 5,527,695).
  • site-specific recombinase system U.S. Pat. No. 5,527,695
  • the FLP/FRT system of yeast the Gin recombinase of phage Mu, the Pin recombinase of E. coli , and the R/RS system of the pSR1 plasmid.
  • the bacteriophage P1 Cre/lox and the yeast FLP/FRT systems constitute two particularly useful systems for site specific integration or excision of transgenes.
  • a recombinase (Cre or FLP) will interact specifically with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequences.
  • the sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT) and therefore, convenient for use with transformation vectors.
  • the FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells.
  • Experiments on the performance of the FLP/FRT system in both maize and rice protoplasts indicate that FRT site structure, and amount of the FLP protein present, affects excision activity. In general, short incomplete FRT sites leads to higher accumulation of excision products than the complete full-length FRT sites.
  • the systems can catalyze both intra- and intermolecular reactions in maize protoplasts, indicating its utility for DNA excision as well as integration reactions.
  • the recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequences is one approach to remedying this situation.
  • the site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event.
  • Cre-lox In the Cre-lox system, discovered in bacteriophage P1, recombination between lox sites occurs in the presence of the Cre recombinase (see, e.g., U.S. Pat. No. 5,658,772, specifically incorporated herein by reference in its entirety). This system has been utilized to excise a gene located between two lox sites which had been introduced into a yeast genome (Sauer, 1987). Cre was expressed from an inducible yeast GAL1 promoter and this Cre gene was located on an autonomously replicating yeast vector.
  • lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule.
  • the deleted DNA segment forms a circular molecule of DNA.
  • the original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites.
  • reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region.
  • ancillary sequences such as selectable marker or reporter genes, for tracking the presence or absence of a desired trait gene transformed into the plant on the DNA construct.
  • ancillary sequences often do not contribute to the desired trait or characteristic conferred by the phenotypic trait gene.
  • Homologous recombination is a method by which introduced sequences may be selectively deleted in transgenic plants.
  • homologous recombination results in genetic rearrangements of transgenes in plants. Repeated DNA sequences have been shown to lead to deletion of a flanked sequence in various dicot species, e.g. Arabidopsis thaliana and Nicotiana tabacum .
  • DSBR double-strand break repair
  • Deletion of sequences by homologous recombination relies upon directly repeated DNA sequences positioned about the region to be excised in which the repeated DNA sequences direct excision utilizing native cellular recombination mechanisms.
  • the first fertile transgenic plants are crossed to produce either hybrid or inbred progeny plants, and from those progeny plants, one or more second fertile transgenic plants are selected which contain a second DNA sequence that has been altered by recombination, preferably resulting in the deletion of the ancillary sequence.
  • the first fertile plant can be either hemizygous or homozygous for the DNA sequence containing the directly repeated DNA which will drive the recombination event.
  • the directly repeated sequences are located 5′ and 3′ to the target sequence in the transgene.
  • the transgene target sequence may be deleted, amplified or otherwise modified within the plant genome.
  • a deletion of the target sequence flanked by the directly repeated sequence will result.
  • DNA sequence mediated alterations of transgene insertions may be produced in somatic cells.
  • recombination occurs in a cultured cell, e.g., callus, and may be selected based on deletion of a negative selectable marker gene, e.g., the periA gene isolated from Burkholderia caryolphilli which encodes a phosphonate ester hydrolase enzyme that catalyzes the hydrolysis of glyceryl glyphosate to the toxic compound glyphosate (U.S. Pat. No. 5,254,801).
  • a negative selectable marker gene e.g., the periA gene isolated from Burkholderia caryolphilli which encodes a phosphonate ester hydrolase enzyme that catalyzes the hydrolysis of glyceryl glyphosate to the toxic compound glyphosate
  • the invention contemplates a transgenic organism comprising within its genome:
  • a first nucleotide sequence encoding a fusion protein comprising a geranyl diphosphate synthase small subunit fused in frame to a geranyl diphosphate synthase large subunit, operatively linked to a first set of expression control sequences that drive expression of the geranyl diphosphate fusion protein in the plant cell;
  • a second nucleotide sequence encoding a limonene synthase, operatively linked to a second set of expression control sequences that drive expression of the limonene synthase in the plant cell;
  • fusion protein and limonene synthase are expressed primarily in the plant cell plastids.
  • the invention contemplates a transgenic organism comprising within its genome:
  • a first nucleotide sequence encoding a fusion protein comprising a geranyl diphosphate synthase small subunit or a geranyl diphosphate synthase large subunit fused in frame to a limonene synthase, operatively linked to a first set of expression control sequences that drive expression of the geranyl diphosphate fusion protein in the plant cell;
  • fusion protein is expressed primarily in the plant cell plastids.
  • the transgenic organisms therefore contain one or more DNA constructs as defined herein as a part of the organism, the DNA constructs having been introduced by transformation of the organism.
  • the geranyl diphosphate synthase small subunit comprises an amino acid sequence selected from Table D1.
  • the geranyl diphosphate synthase large subunit comprises an amino acid sequence selected from Table D2.
  • the limonene synthase comprises an amino acid sequence selected from Table D3.
  • transgenic organisms are characterized by having a terpene content which is at least about 10% higher, at least about 20% higher, at least about 30% higher, at least about 40% higher, at least about 60% higher, at least about 80% higher, or at least about 100% higher than corresponding wild type organism.
  • transgenic organisms are characterized by having a monoterpene content of at least 1 mg/g dry weight, or about 1.2 mg/g dry weight, or about 1.4 mg/g dry weight, or about 1.6 mg/g dry weight, or about 1.8 mg/g dry weight, or about 2.0 mg/g dry weight, or greater then about or about 2.0 mg/g dry weight of seed.
  • the monoterpene produced is selected from the group consisting of limonene, gamma-terpinene and alpha phellandrene, p-cymene, ascaridole and pulegone. In some embodiments the monoterpene is primarily limonene. In some embodiments the monoterpene is a mixture of any of the monoterpenes disclosed herein.
  • transgenic organism will be grown using standard growth conditions as disclosed in the Examples, and compared to the equivalent wild type species.
  • the transgenic organism is a plant.
  • the plant naturally produces a terpene.
  • the transgenic plant is from the genus Camelina .
  • the transgenic plant is selected from Camelina alyssum, Canelina microcarpa, Camelina runelica and Camelina sativa.
  • the geranyl diphosphate synthase fusion protein and limonene synthase are expressed primarily in the seed tissue of the transgenic plant.
  • the term “primarily” means that the relative expression of these proteins is at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% higher in the seed tissue (on a dry weight by dry weight basis) compared to any other plant tissue, in the mature full developed plant, when grown under standard growth conditions.
  • the transgenic plant further expresses an auxillary enzyme as listed in Table D4.
  • Wild-type Camelina sativa was grown in the green house at Donald Danforth Plant Science Center. Peppermint Mentha piperita leaves were harvested from a garden in St. Louis, Mo. in September, 2009.
  • Geranyl diphosphate synthase small subunit without predicted chloroplast transit peptide has been cloned from the peppermint cDNAs with primers: GSSfC and GSSr4 (Table E1) ( FIG. 10 ).
  • Geranyl diphosphate synthase large subunit without predicted chloroplast transit peptide has been cloned from the peppermint cDNAs with primers: GSLfC and GSLr2 ( FIG. 11 ).
  • Geranyl diphosphate synthase (GDS) fusion protein was generated by a 2-step PCR method (Burke et al. 2004 , Arch. Biochem. Biophys., 422, 52-60. Ho et al.
  • LS without predicted chloroplast transit peptide was amplified by PCR using the entire LS clone as template with primers: fwdLSfC3 and LSr ( FIG. 14 ).
  • the resulting GDS and LS PCR fragments were inserted into an NdeI/NotI-digested pET28a expression vector (Novagen) and sequenced, yielding pET28-GDS ( FIG. 12 ) and pET28-LS ( FIG. 14 ), respectively. Both enzyme activities were detected from partially purified E. coli recombinant proteins.
  • pNapin Two E. coli plasmid vectors, pNapin ( FIG. 15 ) and pABC were obtained from Dr. Jaworski (DDPSC). To insert MluI site, pNapin was digested by SacI and ligated with oligo nucleotides: fwdSacIMluISacI and revSacIMluISacI, (Table E1) yielding pNaMluI ( FIG. 16 ).
  • soybean oleosin promoter and soybean oleosin terminator were amplified by the 2-step PCR method using pABC as template with primers: fwdMluIOP, revNotIBamHINdeIOP, fwdNdeIBamHINotIOT and revMluIOT (Table E1).
  • the resulting promoter/terminator fusion DNA fragment was inserted into MluI-digested pNaMluI ( FIG. 16 ) and sequenced, yielding pNaMluIOleosin ( FIG. 17 ).
  • Rapeseed napin promoter and soybean glycinin terminator were amplified by the 2-step PCR method using pNapin as template with primers: fwdAscINP, revNotIBamHINdeINP, fwdNdeIBamHINotIGT and revAscIGT (Table E1).
  • the resulting napin promoter/glycinin terminator fusion DNA fragment was inserted into AscI-digested pNapin and sequenced, yielding pNaAscINapin ( FIG. 18 ).
  • the entire coding sequence of RuBisCO small subunit has been cloned from sweet pea siliques with primers: RuSfwd and RuSrev (Table E1) ( FIG. 19 ).
  • the RuBisCO small subunit transit peptide was amplified by PCR using the entire RuBisCO clone as template with primers: RuSfwd and revBamHIRuTP (Table E1).
  • the resulting PCR product was inserted into NdeI/BamHI-digested pNaMluIOleosin ( FIG. 17 ) and pNaAscINapin ( FIG. 18 ), and sequenced, yielding two entry vectors: pNaMluIOleosinTP ( FIG. 20 ) and pNaAscINapinTP ( FIG. 21 ), respectively.
  • the pNaMluIOleosinTP vector FIG.
  • the pNaAscINapinTP vector ( FIG. 21 ) contains AscI, the napin promoter, the transit peptide, BamHI, NotI, the glycinin terminator and AscI in this order.
  • the GDS sequence with BamHI/NotI sites was amplified by PCR using pET28-GDS ( FIG. 12 ) as template with primers: fwdBamHIGDS and GSLr2 (Table E1).
  • the resulting PCR product was inserted into BamHI/NotI-digested pNaMluIOleosinTP ( FIG. 20 ), yielding pNaMluIOleosinTPGDS ( FIG. 22 ).
  • the LS sequence with BamHI/NotI sites was amplified by PCR using pET28-LS ( FIG. 14 ) as template with primers: fwdBamHILS and LSr.
  • the resulting PCR product was inserted into BamHI/NotI digested pNaAscINapinTP ( FIG. 21 ), yielding pNaAscINapinTPLS ( FIG. 23 ).
  • a pRS binary vector was obtained from Dr. Jan Jaworski, which contains a Discosoma red fluorescent protein (DsRed) as a selection marker, and AscI/MluI restriction enzyme sites between the left border and right border T-DNA repeat sequences.
  • DsRed Discosoma red fluorescent protein
  • AscI/MluI restriction enzyme sites between the left border and right border T-DNA repeat sequences.
  • pRS was digested by BamHI/HindIII and ligated with oligo nucleotides: fwdBamHIEcoRIHindIII and revHindIIIEcoRIBamHI (Table E1), yielding pRSe2 ( FIG. 24 ). MluI-digested GDS from pNaMluIOleosinTPGDS ( FIG.
  • GDSLS GDSLS Camelina transformation vector for cytosolic expression was prepared with pNaMluIOleosin ( FIG. 17 ) and pNaAscINapin ( FIG. 18 ). These entry vectors differ only in the absence of the sequence for TP from pNaMluIOleosinTP ( FIG. 20 ) and pNaAscINapinTP ( FIG. 21 ), respectively. NdeI/NotI-digested GDS and LS from the pET28-GDS ( FIG. 12 ) and pET28-LS ( FIG. 14 ) were inserted into pNaMluIOleosin ( FIG. 17 ) and pNaAscINalin ( FIG.
  • GDSLS binary vector
  • GDS9aaLS nucleotide sequence with NdeI/NotI sites was amplified by the 2-step PCR method using the TPGDSTPLS plasmid ( FIG. 25 ) as template with primers: GSSfC, rev9aaGSLr2, fwd9aaLSfC3 and LSr (Table E1).
  • the resulting PCR product was inserted into NdeI/NotI-digested pET28 ( FIG. 29 ), yielding pET28-GDS9aaLS ( FIG. 30 ).
  • LS9aaGDS nucleotide sequence with NdeI/NotI sites was amplified by the 2-step PCR method using the TPGDSTPLS plasmid ( FIG.
  • the TPGDSTPLS vector ( FIG. 25 ) and the GDSLS vector ( FIG. 28 ) were transformed into Agrobacterium tumefaciens strain GV3101 (pMP90) using a freeze-thaw method (Weigel and Glazebrook (2006) Cold Spring Harb Protoc ., doi:10.1101/pdb.prot4666). Selection of transformed bacteria was carried out on YEP medium containing 10 g/l peptone, 5 g/l yeast extract and 5 g/l NaCl at pH 6.8 with 25 mg/l rifampicin, 40 mg/l gentamicin and 50 mg/l kanamycin.
  • Overnight culture of the transformed bacteria was transferred into 2 l flask containing 300 ml YEP medium with 50 mg/l kanamycin and incubated at 28° C. for 24 hours.
  • Cells were harvested by centrifugation for 20 min at root temperature at 5000 g and then resuspended in an infiltration medium containing half strength Murashige and Skoog Basal Medium, 50 g/l sucrose and 0.05% (v/v) Silwet L77 (Lehle Seeds, Round Rock, Tex.) to a final OD 600 of between 1.0 to 1.5.
  • Camelina transformation was performed using a floral dip method (Lu and Kang (2008) Plant Cell Rep., 27, 273-8.). Camelina plants were inoculated with the Agrobacterium suspension prepared as described above. A flowering Camelina plant was placed into a vacuum desiccator and the inflorescences were immersed into the suspension in a 500 ml beaker. The suspension with the inflorescences was degassed under vacuum for 5 min. The inoculated plants were covered with plastic trays for 24 hours before returned to normal growth in greenhouse. Transgenic fluorescent mature seeds were illuminated by a green LED flashlight, and visually detected using a red-lens screen.
  • the expressed and purified E. coli recombinant GDS, GSL, GSS and LS were observed on SDS-PAGE ( FIG. 3A ). Functional activity of the GDS protein was detected using isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to produce geranyl diphosphate (GPP) which was hydrolyzed to geraniol ( FIG. 3B ). Also, functional activity of the LS protein was detected using GPP as substrate to produce limonene ( FIG. 3C ). GC-MS separated limonene, three hydrocarbons (C 10 H 16 ) and two oxidized monoterpenes (C 10 H 16 O, C 10 H 14 O) from the T2 seeds ( FIG. 4 ). Limonene constituted 97.3% of the total monoterpenes calculated from the signal intensities.
  • IPP isopentenyl diphosphate
  • DMAPP dimethylallyl diphosphate
  • GPP geranyl diphosphat
  • the limonene content of the T3 homozygous seeds ranged from 1.8 to 3 mg/g seeds ( FIG. 5 ).
  • T-DNA insertion was confirmed by PCR analyses of total DNA of T2 leaves ( FIG. 6 ). Expression of mRNA from the integrated DsRed, GDS and LS were analyzed by RT-PCR ( FIG. 7 ). Both GDS and LS enzyme activities were detected from T2 seeds in vitro by a coupling enzyme assay ( FIG. 8 ). A reaction mixture containing transgenic seed extract catalyzed the enzymatic reactions of GDS and LS, i.e. producing limonene from IPP and DMAPP.
  • GDS9aaLS and LS9aaGDS enzymes were expressed in E. coli host cells BL21(DE3)RIL containing pET28-GDS9aaLS ( FIG. 30 ) and pET28-LS9aaGDS ( FIG. 31 ). Both recombinant proteins were separated by SDS-PAGE and detected by Sypro-Ruby staining (Invitrogen) ( FIG. 9A ).
  • Functional activity of the fusion GDS9aaLS protein was not detected using isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to produce limonene ( FIG. 9B ).
  • Functional activity of the fusion LS9aaGDS protein was detected using isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to produce limonene ( FIG. 9C ).
  • This example describes the biosynthesis and accumulation of the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon 5-epi-aristolochene in camelina seed by expressing appropriate combinations of terpene biosynthetic enzymes.
  • biosynthetically appropriate combination of enzymes refers to a combination of terpene biosynthetic enzymes that facilitates the biosynthesis of a monoterpene or sesquiterpene of interest. Such combinations include a combination of: 1) a geranyl diphosphate synthase and a monoterpene synthase that catalyzes the formation of a monoterpene of interest, or 2) a combination of a farnesyl diphosphate synthase and a sesquiterpene synthase that catalyzes the formation of a sesquiterprene of interest.
  • a biosynthetically appropriate combination of nucleotide sequences refers to nucleotide sequences that encode such biosynthetically appropriate combinations of enzymes.
  • Geranyl diphosphate synthase (peppermint) (Burke C. C., Wildung M. R. and Croteau R. (1999) Proc Natl Acad Sci USA., 96, 13062-7) and (4S)-limonene synthase (peppermint) (Colby, S. M. Alonso, W. R., Katahira, E. J., McGarvey, D. J. & Croteau, R. J. Biol. Chem. 268, 23016-23024 (1993)) as well as farnesyl diphosphate synthase ( arabidopsis ) (Cunillera, N. et al. J. Biol. Chem.
  • seeds contain a variety of other plastids as well, including, for example, proplastids, etioplasts, chromoplasts, leucoplasts, amyloplasts, and photoheterotrophic plastids. Consequently, plastid transit peptides that target peptides, polypeptides, or proteins to any of these types of plastids in seeds can also be employed in the methods of the present invention.
  • the geranyl diphosphate synthase from peppermint is a heterodimer, which was expressed as a fusion protein in camelina seed.
  • Each reading frame was placed under the control of either the oleosin (Rowley et al. Biochim. Biophys. Acta 1345, 1-4 (1997)), napin (Josefsson et al. J. Biol. Chem. 262, 12196-12201 (1987)), or glycinin (Nielsen et al. Plant Cell 1, 313-328 (1989)) promoter; any given promoter was used only once in an expression vector to avoid potential gene silencing.
  • the effect of overexpression of the DXS Estévez et al. Plant Physiol.
  • transgenic events were achieved and analyzed with each vector construct tested. Typically, fifteen plants were transformed with each expression vector construct. A total of ca. 70-140 DsRed-positive seeds were obtained, representing 0.2-0.8% of total seeds produced in fifteen plants.
  • Initial GC-MS analyses were carried out on extracts of individual Ti seeds. In subsequent generations, ten transgenic (red) seeds from each plant were combined for terpene extraction with subsequent GC-MS analysis. Only seeds from the T3 generation that were homozygous lines (produced >95% red seeds) were used for further analysis.
  • transgenic camelina seed higher accumulation levels of (4S)-limonene were achieved in plastid and higher accumulation levels of 5-epi-aristolochene were also achieved in plastid.
  • the activities of both the prenyltransferases and terpene synthases were tested in vitro in crude protein extracts prepared from camelina seed in order to examine whether the differences in terpene accumulation in the plastid and cytosol experiments were due to variations in enzyme activity. Both the plastidic and cytosolic accumulation experiments yielded similar ranges of GDS specific activity (TPGDS TPLS, 7-13.5 pmol min ⁇ 1 mg ⁇ 1 protein; GDS LS, 6.5-15 pmol min ⁇ 1 mg ⁇ 1 protein; FIG.
  • TPGDS TPLS 0.9 pmol min ⁇ 1 mg ⁇ 1 protein
  • TPGDS TPLS DXS 1.1 pmol min ⁇ 1 mg ⁇ 1 protein
  • Fusions between prenyltransferases and terpene synthase occur in nature, at least for formation of the diterpenes, the fusicoccins, in the plant pathogenic fungus Phomopsis amygdali (Toyomasu et al. Proc. Natl. Acad. Sci. USA 104, 3084-3088 (2007)). Fusion of farnesyl diphosphate synthase from Artemisia annua and 5-epi-aristolochene synthase from tobacco produced a functional chimera in E. coli .
  • the K m values were unchanged in the fusion protein when compared to the individual enzymes, however, a more efficient conversion of IPP to 5-epi-aristolochene was achieved with the fusion protein (Brodelius et al. Eur. J. Biochem. 269, 3570-3577 (2002)).
  • the geranyl diphosphate synthase expressed herein in camelina seed was a fusion of heteromonomers (Burke et al. Arch. Biochem. Biophys. 422, 52-60 (2004)).
  • Specialized cellular compartments have evolved to store terpenes in plants, such as the subcuticular space between trichome head cells and the cuticle that encloses them in herbaceous plant species (Gershenzon et al. Anal. Biochem. 200, 130-138 (1992)). Due to the high volatility of monoterpenes and the lack of a specialized storage compartment in camelina seed, head-space analysis of developing seed and mature, stored seed was carried out on (4S)-limonene-accumulating transgenic camelina to estimate yield loss due to release to the atmosphere.
  • camelina seed is a suitable synthetic biology platform for the production and accumulation of cyclic hydrocarbons that can function as components of biofuels.
  • the plant is genetically tractable by floral dip, selection of transgenic seed is facilitated by florescence resulting from expression of the gene encoding DsRed in the transformation vector, and transgene expression is stable over at least generations.
  • loss of volatile terpenes during seed development and storage is minimal, and acid hydrolysis of terpene O-glucosides that are formed results in aromatic derivatives.
  • Cyclic terpenes are currently being considered as alternatives to diesel (Peralta-Yahya et al. Nat. Commun. 2, Article 483 (2011)).
  • cyclic terpene hydrocarbons can be stably over-produced and accumulated in an oilseed.
  • we expect to increase terpene accumulation by optimizing flux through the biochemical pathway by altering gene dosage of prenyltransferase vs. terpene synthase to balance the difference in steady-state kinetics between these two classes of enzymes.
  • Geranyl diphosphate synthase small subunit (GSS) and geranyl diphosphate synthase large subunit (GSL) have been cloned from the peppermint cDNAs with primers: GSSfC/GSSr4 and GSLfC/GSLr2, respectively (see Table E1 for primer sequences).
  • GDS geranyl diphosphate synthase fusion protein
  • Limonene synthase (LS) has been cloned from the peppermint cDNAs with primers: LSuf/LSr and fwdLSfC3/LSr.
  • Farnesyl diphosphate synthase (FDS) gene has been cloned from the Arabidopsis cDNA with primers: FDSf/FDSr.
  • the 5-epi-aristolochene synthase (EAS) sequence was amplified from the tobacco DNA with primers: fwdBamHIEAS/NtEASrc.
  • the pNaMluIOleosin entry vector contains a soybean oleosin promoter (OP) and a soybean oleosin terminator (OT).
  • the pNaAscINapin entry vector contains a rapeseed napin promoter (NP) and a soybean glycinin terminator (GT).
  • a binary vector, pRS was a kind gift from Dr. Jan Jaworski (Donald Danforth Plant Science Center, MO).
  • the nucleotide sequence was modified as follows:
  • the pRSe2 vector contains a Discosoma red fluorescent protein (DsRed) as a selection marker between the left and right border T-DNA repeat sequences.
  • DsRed Discosoma red fluorescent protein
  • the pea Rubisco small subunit transit peptide (TP) was inserted into pNaMluIOleosin and pNaAscINapin, yielding pNaMluIOleosinTP (OP-TP-OT) and pNaAscINapinTP (NP-TP-GT) entry vectors, respectively.
  • TP pea Rubisco small subunit transit peptide
  • OP-TP-OT pNaMluIOleosinTP
  • NP-TP-GT pNaAscINapinTP
  • the nucleotide sequences of OP-TP-GDS-OT and NP-TP-LS-GT, OP-GDS-OT and NP-LS-GT, OP-FDS-OT and NP-EAS-GT, and OP-TP-FDS-OT and NP-TP-EAS-GT were inserted into pRSe2 and sequenced, yielding TPGDS TPLS (plastid), GDS LS (cytosol), FDS EAS (cytosol), and TPFDS TPEAS (plastid) camelina transformation vectors, respectively.
  • Fusion proteins of GDS and LS were constructed, which contained a nine amino acid linker (9aa, SGGSGGSGG (SEQ ID NO:35).
  • the nucleotide sequences of OP-GDS-9aa-LS-OT, OP-TP-GDS-9aa-LS-OT, and OP-TP-LS-9aa-GDS-OT were inserted into pRSe2 and sequenced, yielding GDSLS fusion (cytosol), TPGDSLS fusion (plastid), TPLSGDS fusion (plastid) camelina transformation vectors, respectively.
  • the Arabidopsis DXS coding sequence with its own transit peptide was a kind gift from Dr. Ed Cahoon (University of Kansas-Lincoln, Nebr.).
  • pRSDXS DXS expression is controlled by a soybean glycinin promoter.
  • the nucleotide sequences of OP-TP-GDS-OT and NP-TP-LS-GT were inserted into pRSDXS and sequenced, yielding a TPGDS TPLS DXS (plastid) camelina transformation vector.
  • the generated transformation vectors were transformed into Agrobacterium tumefaciens strain GV3101 (pMP90) using a freeze-thaw method (Weigel et al. CSH Protoc . doi:10.1101/pdb.prot4666 (2006)). Selection of transformed bacteria was carried out on YEP medium containing 10 gl ⁇ 1 Bacto-peptone, 5 gl ⁇ 1 yeast extract and 5 gl ⁇ 1 NaCl at pH 6.8 with 25 mgl ⁇ 1 rifampicin, 40 mgl ⁇ 1 gentamicin and 50 mgl ⁇ 1 kanamycin. The plasmid insertion was confirmed by PCR.
  • Wild-type camelina plant was grown in the Donald Danforth Plant Science Center green house. Camelina transformation was performed using a floral dip method (Lu et al. Plant Cell Rep. 27, 273-278 (2008)). Camelina plants were inoculated with the Agrobacterium suspension prepared as described above. One or two flowering camelina plants were placed into a vacuum desiccator and the inflorescences were immersed into the suspension in a 500 ml beaker. The suspension with the inflorescences was degassed under vacuum for 5 min. The inoculated plants were covered with plastic trays for 24 hours before returning to the greenhouse. Mature seeds of the transformed plants were illuminated with a green LED flashlight, and transgenic seeds identified based on their fluorescence visualized by a red-lens screen.
  • Oven temperature for (4S)-limonene analysis was 50° C. for 3 min, raised to 80° C. at a rate of 10° C. min ⁇ 1 , held for 3 min, raised again to 300° C. at 40° C. min ⁇ 1 , and held for 3 min.
  • Oven temperature for 5-epi-aristolochene analysis was 50° C. for 3 min, raised to 180° C. at a rate of 5° C. min ⁇ 1 , raised to 300° C. at 40° C. min ⁇ 1 , and held for 3 min.
  • the (4S)-limonene and 5-epi-aristolochene concentrations were calculated using (4S)-limonene and valencene as standards (Sigma), respectively. Other monoterpenes and sesquiterpenes were assigned by comparison of their EI-MS spectra with those of the NIST library.
  • Total protein extract was prepared from dry mature seeds. Ca. 22 seeds (corresponding to ⁇ 20 mg) were ground in a 1.5 ml tube with a plastic homogenizer on ice for 3 min in 20 ⁇ l mg ⁇ 1 of extraction buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 5 mM 2-mercaptoethanol and a protease inhibitor cocktail (Sigma). The protein extract was centrifuged at 15000 g for 30 min at 4° C. twice. 45 ⁇ l aliquots of the resulting supernatant were frozen in liquid nitrogen and stored at ⁇ 80° C.
  • the enzyme activity of GDS was analyzed as follows: reactions were performed in a total volume of 1 ml adjusted to 50 mM Hepes pH 7.2, 10% (v/v) glycerol, 20 mM MgCl 2 , 0.5 mM DTT and with 50 ⁇ g E. coli recombinant (4S)-limonene synthase purified from pET28-LS BL21(DE3)RIL, 2 nmol IPP, 2 nmol DMAPP and 45 ⁇ l camelina seed protein extract.
  • the enzyme reaction was initiated by the addition of IPP and DMAPP, overlaid with hexane and incubated at 30° C. for 30 min to 4 hr.
  • the enzyme activity of LS was analyzed as follows: reactions were performed in a total volume of 1 ml adjusted to 50 mM Hepes pH 7.2, 10% (v/v) glycerol, 20 mM MgCl 2 , 0.5 mM DTT, 500 mM KCl and with 2 nmol GPP and 45 ⁇ l camelina seed protein extract.
  • the enzyme reaction was initiated by the addition of GPP, overlaid with hexane and incubated at 30° C. for 1 hr to 8 hr. The reaction was stopped by chilling on ice followed by vigorous mixing. After adding the internal standard, enzymatically produced (4S)-limonene was extracted with hexane 3 times. The combined hexane extract was dehydrated by Na 2 SO 4 , concentrated and analyzed by GC-MS.
  • the (4S)-limonene content was measured from the peak height of an ion of m/z 136.
  • the volatile emission of mature seed on storage was also monitored (number of seeds produced per plant was ca. 1700).
  • the total (4S)-limonene emission during seed development was estimated by integration of each time point from two individual cultivation periods.
  • glycosides were identified with precursor ion scan (m/z 161.0) and product ion scan (m/z 391.2) in negative ionization mode.
  • the quantification was performed using phenyl-B-D-glucopyranoside as an internal standard by multiple reactions monitoring (MRM) scan.

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Publication number Priority date Publication date Assignee Title
CN104988140A (zh) * 2015-05-20 2015-10-21 中国科学院华南植物园 一种来源于水稻的启动子及其应用
US20220256794A1 (en) * 2019-05-08 2022-08-18 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization Terpene synthases and transporters

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CN103898037B (zh) * 2014-03-11 2017-11-17 中国科学院青岛生物能源与过程研究所 一种联产香叶醇和橙花醇的基因工程菌及其构建方法和应用
SG11201703132UA (en) * 2014-10-22 2017-05-30 Temasek Life Sciences Lab Ltd Terpene synthases from ylang ylang (cananga odorata var. fruticosa)
WO2020033705A2 (fr) * 2018-08-08 2020-02-13 Board Of Trustees Of Michigan State University Production améliorée de terpénoïdes à l'aide d'enzymes ancrées à des protéines de surface de gouttelettes lipidiques
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291745B1 (en) * 1993-04-02 2001-09-18 Pioneer Hi-Bred International, Inc. Limonene and other downstream metabolites of geranyl pyrophosphate for insect control in plants
US20080201796A1 (en) 2005-07-01 2008-08-21 University Of Kentucky Research Foundation Transformed plants accumulating mono-and/or sesquiterpenes
US20080281135A1 (en) 2005-01-27 2008-11-13 Librophyt System For Producing Terpenoids In Plants
US20090123984A1 (en) 2005-04-19 2009-05-14 The University Of Kentucky Research Foundation Transformed plants accumulating terpenes
US20100138954A1 (en) 2007-04-03 2010-06-03 Philip Morris Products S.A. Genes Encoding Z,Z-Farnesyl Diphosphate Synthase and A Sesquiterpene Synthase with Multiple Products and Uses Thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999002030A1 (fr) * 1997-07-11 1999-01-21 Washington State University Research Foundation MONOTERPENES SYNTHASES TIREES DE SAPIN GRANDISSIME (Abies grandis)
EP2406378A4 (fr) * 2009-03-11 2013-04-24 Sapphire Energy Inc Production de biocarburant dans des procaryotes et eucaryotes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291745B1 (en) * 1993-04-02 2001-09-18 Pioneer Hi-Bred International, Inc. Limonene and other downstream metabolites of geranyl pyrophosphate for insect control in plants
US20080281135A1 (en) 2005-01-27 2008-11-13 Librophyt System For Producing Terpenoids In Plants
US20090123984A1 (en) 2005-04-19 2009-05-14 The University Of Kentucky Research Foundation Transformed plants accumulating terpenes
US20080201796A1 (en) 2005-07-01 2008-08-21 University Of Kentucky Research Foundation Transformed plants accumulating mono-and/or sesquiterpenes
US20100138954A1 (en) 2007-04-03 2010-06-03 Philip Morris Products S.A. Genes Encoding Z,Z-Farnesyl Diphosphate Synthase and A Sesquiterpene Synthase with Multiple Products and Uses Thereof

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
Augustin, Jörg M., et al. "Production of mono-and sesquiterpenes in Camelina sativa oilseed." Planta 242.3 (2015): 693-708. *
Blank, I., and W. Grosch. "Evaluation of potent odorants in dill seed and dill herb (Anethum graveolens L.) by aroma extract dilution analysis." Journal of food science 56.1 (1991): 63-67. *
Buttery, Ron G., Louisa C. Ling, and Bock G. Chan. "Volatiles of corn kernels and husks: Possible corn ear worm attractants." Journal of Agricultural and Food Chemistry 26.4 (1978): 866-869. *
Charles, Denys J., and James E. Simon. "Comparison of extraction methods for the rapid determination of essential oil content and composition of basil." Journal of the American Society for Horticultural Science 115.3 (1990): 458-462. *
Emanuelsson O, Nielsen H, von Heijne G., 1999, ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites Protein Sci. 8(5):978-84. *
International Search Report for PCT/US2013/040791.
Le, Brandon H., et al. "Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors." Proceedings of the National Academy of Sciences 107.18 (2010): 8063-8070. *
Morris, Wayne L., et al. "Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle." Journal of Experimental Botany 57.12 (2006): 3007-3018. *
Okada, Kazunori, et al. "Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis." Plant physiology 122.4 (2000): 1045-1056. *
Rodriguez-Concepción, Manuel, and Albert Boronat. "Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics." Plant physiology 130.3 (2002): 1079-1089. *
van Wijk, K. J., and S. Baginsky, 2011. "Plastid proteomics in higher plants: current state and future goals." Plant physiology 155.(4): 1578-1588. *
Wu, Shuiqin, et al. "Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants." Nature biotechnology 24.11 (2006): 1441-1447. *
Zavala-Páramo, Guadalupe, et al. "Isolation of an elicitor-stimulated 5-epi-aristolochene synthase gene (gPEAS1) from chili pepper (Capsicum annuum)." Physiologia Plantarum 110.3 (2000): 410-418. *

Cited By (2)

* Cited by examiner, † Cited by third party
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CN104988140A (zh) * 2015-05-20 2015-10-21 中国科学院华南植物园 一种来源于水稻的启动子及其应用
US20220256794A1 (en) * 2019-05-08 2022-08-18 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization Terpene synthases and transporters

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